Multikinase Inhibitor Regorafenib Research Articles

Circulating microRNA Analysis in a Prospective Co-clinical Trial Identifies MIR652-3p as a Response Biomarker and Driver of Regorafenib Resistance Mechanisms in Colorectal Cancer.

The multi-kinase inhibitor (mKi) regorafenib has demonstrated efficacy in chemorefractory patients with metastatic colorectal cancer (mCRC). However, lack of predictive biomarkers and concerns over significant toxicities hamper the use of regorafenib in clinical practice. Serial liquid biopsies were obtained at baseline and monthly until disease progression in chemorefractory patients with mCRC treated with regorafenib in a phase II clinical trial (PROSPECT-R n = 40; NCT03010722) and in a multicentric validation cohort (n = 241). Tissue biopsies collected at baseline, after 2 months and at progression in the PROSPECT-R trial were used to establish patient-derived organoids (PDO) and for molecular analyses. MicroRNA profiling was performed on baseline bloods using the NanoString nCounter platform and results were validated by digital-droplet PCR and/or ISH in paired liquid and tissue biopsies. PDOs co-cultures and PDO-xenotransplants were generated for functional analyses. Large-scale microRNA expression analysis in longitudinal matched liquid and tissue biopsies from the PROSPECT-R trial identified MIR652-3p as a biomarker of clinical benefit to regorafenib. These findings were confirmed in an independent validation cohort and in a "control" group of 100 patients treated with lonsurf. Using ex vivo co-culture assays paired with single-cell RNA-sequencing of PDO established pre- and post-treatment, we modeled regorafenib response observed in vivo and in patients, and showed that MIR652-3p controls resistance to regorafenib by impairing regorafenib-induced lethal autophagy and by orchestrating the switch from neo-angiogenesis to vessel co-option. Our results identify MIR652-3p as a potential biomarker and as a driver of cell and non-cell-autonomous mechanisms of resistance to regorafenib.

Abstract B079: Identification of synergistic drug combinations including MEK inhibitor and agents modulating the tumor microenvironment in an electroporation-induced pancreatic cancer model

Abstract Pancreatic ductal adenocarcinoma (PDAC) is refractory to cytostatic and immuno-oncology therapeutic strategies, leaving surgical resection of the tumor as the only treatment that can significantly prolong survival. We are therefore exploring optimized treatment strategies targeting cancer cells as well as infiltrating immune cells and the tumor microenvironment (TME) in an autochthonous mouse model for PDAC. Single focal tumors are induced through in vivo electroporation of gene constructs into the pancreas which encode mutated Kras and facilitate knock out the tumor suppressor genes P53, Cdkn2a and/or Smad4 via CRISPR/Cas9. The resulting tumors recapitulate the human disease with respect to genetics, histology and TME composition. Furthermore, this model allows the generation of tumors reflecting different PDAC subtypes, as well as the resection of primary tumors in combination with the (neo-)adjuvant regimens. Like the human disease, these tumors are refractory to chemotherapy and immune checkpoint blockade, whereas targeted therapy by means of MEK inhibitors merely leads to temporary delay of tumor outgrowth. We therefore tested the combination of MEK inhibitor treatment with different, potentially complementary modalities. We found the combination with agonist anti-CD40 antibodies to be highly effective. Prominent regressions are observed, and in combination with primary tumor resection long-term complete responses can be achieved even in a metastatic variant of the model. Apart from the direct cytostatic impact of MEKi on the Kras-transformed tumor cells, the mechanism of action of this synergistic drug combination involves enhancement of the anti-tumor T-cell response, suppression of CD4+ Tregs, and reduction of MDSCs and M2-type macrophages in favor of M1-type pro-inflammatory macrophages. A second highly effective regimen as identified in our experiments involves the combination of MEK inhibitors with the multi-kinase inhibitor regorafenib. In contrast to the aforementioned regimen, the complementary action of these drugs involves modulation of the TME by regorafenib. While the clinical translation of the MEKi/CD40 Ab regimen is hampered by toxicity issues encountered with agonist anti-CD40 Ab, the synergy as observed in the MEKi/regorafenib regiment provides a tangible path towards clinical testing. Citation Format: Aline S. Konrad, Tanja Hägele, Stefan Zens, Caroline Vent, Moritz Hamberger, Sarah Baiker, Aron Maes, Carl-Stephan Leonhardt, Oliver Strobel, Oliver Politz, Dieter Zopf, Daniel Baumann, Julian Mochayedi, Rienk Offringa. Identification of synergistic drug combinations including MEK inhibitor and agents modulating the tumor microenvironment in an electroporation-induced pancreatic cancer model [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B079.

Regorafenib and glioblastoma: a literature review of preclinical studies, molecular mechanisms and clinical effectiveness.

Glioblastoma IDH wild type (GBM) is a very aggressive brain tumour, characterised by an infiltrative growth pattern and by a prominent neoangiogenesis. Its prognosis is unfortunately dismal, and the median overall survival of GBM patients is short (15 months). Clinical management is based on bulk tumour removal and standard chemoradiation with the alkylating drug temozolomide, but the tumour invariably recurs leading to patient's death. Clinical options for GBM patients remained unaltered for almost two decades until the encouraging results obtained by the phase II REGOMA trial allowed the introduction of the multikinase inhibitor regorafenib as a preferred regimen in relapsed GBM treatment by the National Comprehensive Cancer Network (NCCN) 2020 Guideline. Regorafenib, a sorafenib derivative, targets kinases associated with angiogenesis (VEGFR 1-3), as well as oncogenesis (c-KIT, RET, FGFR) and stromal kinases (FGFR, PDGFR-b). It was already approved for metastatic colorectal cancers and hepatocellular carcinomas. The aim of the present review is to focus on both the molecular and clinical knowledge collected in these first three years of regorafenib use in GBM.

CTIM-32. A PARALLEL COHORT PHASE 2 STUDY OF REGORAFENIB PLUS NIVOLUMAB FOR RECURRENT OR METASTATIC SOLID TUMORS: RESULTS IN PATIENTS WITH GLIOBLASTOMA MULTIFORME (GBM) OR ANAPLASTIC ASTROCYTOMA (AA)

Abstract INTRODUCTION The multikinase inhibitor regorafenib improved overall survival (OS) versus lomustine in relapsed GBM in the randomized phase 2 REGOMA study. The aim of this study was to evaluate the efficacy of regorafenib combined with nivolumab in this setting (NCT04704154). METHODS Patients aged ≥ 18 years with recurrent GBM/AA (WHO 2016 classification) after radiotherapy plus temozolomide, and an ECOG performance status (PS) of 0/1, received oral regorafenib 90 mg/day on days 1–21 and intravenous nivolumab 480 mg on day 1 of each 28-day cycle. If tolerated, the regorafenib dose could be escalated to 120 mg/day from cycle 2. Prior regorafenib treatment or immunotherapy was not allowed. The primary endpoint was objective response rate (ORR) using RANO criteria. Secondary endpoints included progression-free survival (PFS), OS, and safety (MedDRA; CTCAE v5.0). RESULTS Of the 36 patients enrolled, 30 were treated and were eligible for analysis. Median age was 60 years, 63% had an ECOG PS of 1, and 90% had GBM (10% AA). All patients discontinued treatment as of the primary analysis (March 9, 2023). One patient had a best-overall partial response (ORR; 3%) and nine (30%) had stable disease. Median number of cycles of regorafenib and nivolumab infusions was three. Median PFS (RANO) was 2.7 months (80% CI 1.8, 7.3) and 6-month PFS was 31.5%. Median OS was 8.0 months (80% CI 4.6, 12.4) and 12-month OS was 33.7%. Thirteen patients (43%) had grade 3/4 treatment-emergent adverse events (TEAEs), which were drug-related in 12 patients (40%). The most common drug-related grade 3/4 TEAEs were AST increase (13%), ALT increase (10%), and hypertension (10%). There were no immune-mediated serious TEAEs or drug-related grade 5 TEAEs. CONCLUSION The efficacy results of this study do not support further evaluation of regorafenib combined with nivolumab in GBM/AA. The safety profile was acceptable and manageable.

BIOM-13. ASSOCIATION OF HAND-FOOT SKIN REACTIONS WITH SURVIVAL IN RECURRENT GLIOBLASTOMA PATIENTS TREATED WITH REGORAFENIB

Abstract INTRODUCTION The phase 2 REGOMA trial suggested a survival benefit of the multikinase inhibitor regorafenib for treating patients with glioblastoma at first recurrence. Hand-foot skin reactions (HFSR) following multikinase inhibitor therapy have been reported to predict prolonged overall survival (OS) in patients with solid tumors. The predictive value of HFSR in patients with recurrent glioblastoma undergoing regorafenib therapy remains to be investigated. METHODS Fifty-seven patients (age range, 35-77 years) with recurrent IDH-wildtype glioblastomas were retrospectively identified from five centers in Germany and Switzerland. Regorafenib was administered at 160 mg/d for the first three weeks of each 4-week cycle, with individual dose adjustments according to toxicity. Patients were stratified into two groups based on the occurrence of HFSR following regorafenib. Group differences were investigated using the Mann-Whitney rank-sum and Fisher's exact tests. Kaplan-Meier analysis was used for univariate analyses, and the Cox proportional hazards regression model for multivariate analyses. RESULTS Patients received a median of two regorafenib cycles (range, 1-14 cycles). Median follow-up after recurrence was 7.0 months (range, 0.6-35.9 months). The median number of previous treatment lines was 2 (range, 1-6 lines). At the time of data analysis, 47 patients (82%) had died. HFSR were observed in 14 out of 57 patients (25%) and associated with a significantly longer OS (10.6 vs. 6.0 months; P = 0.040). No significant group differences (HFSR compared to no HFSR) were observed regarding age, the extent of resection, MGMT promoter methylation, Karnofsky performance status, or the number of completed regorafenib cycles (all P > 0.05). Cox proportional hazards regression analysis confirmed that HFSR were associated with longer OS (P = 0.039; HR, 0.438), independent of age, Karnofsky performance status, and the number of previous treatment lines (all P > 0.05). CONCLUSION Our data suggest that the occurrence of HFSR following regorafenib is associated with longer OS in recurrent glioblastoma patients.

JS09.6.A ASSESSMENT OF RESPONSE TO REGORAFENIB IN RECURRENT GLIOMA PATIENTS USING FET PET AND MRI INCLUDING ADC VALUES

Abstract BACKGROUND The phase 2 REGOMA trial suggested that glioblastoma patients treated with the multikinase inhibitor regorafenib at first progression have significantly longer overall survival than patients treated with lomustine. Here, we evaluated the value of O-(2-[18F]fluoroethyl)-L-tyrosine (FET) PET and MRI for the evaluation of response to regorafenib in recurrent glioma patients. MATERIAL AND METHODS Twenty patients (age range, 30-72 years) with CNS WHO grade 3 or 4 gliomas (glioblastoma, 85%) at recurrence were treated according to the REGOMA trial with regorafenib at 160 mg/d for the first three weeks of each 4-week cycle, with individual dose adjustment according to toxicity. FET PET and MRI were performed at baseline and follow-up after the second cycle. Static FET PET parameters comprised tumor-to-brain ratios (TBR) and metabolic tumor volumes (MTV). Parameters derived from dynamic FET PET acquisition were time-to-peak and slope. MRI response assessment was based on RANO criteria. Additionally, mean and minimum apparent diffusion coefficients (ADC) were obtained from diffusion-weighted MRI. FET PET parameters and ADC thresholds were defined using ROC analyses to predict an overall survival of ≥ 6 months. The predictive values of FET PET parameters, ADC values, and RANO criteria were subsequently evaluated using univariate and multivariate survival estimates. RESULTS Patients received a median of three regorafenib cycles (range, 2-10 cycles). After treatment initiation, the median follow-up was 8.6 months (range, 3.2-27.3 months). The median number of previous treatment lines was two (range, 1-4 lines). At the time of data analysis, 17 patients (85%) had died. After two cycles of regorafenib, a regorafenib-induced reduction of mean TBR values by ≥ 10% predicted a longer overall survival (9.9 vs. 5.3 months; P=0.023). Interestingly, absolute mean TBR values ≤ 2.0 at follow-up were prognostic and associated with longer OS (10.6 vs. 4.5 months; P=0.007). In contrast, MTV, dynamic PET parameters, RANO criteria, and ADC values were not predictive for response or prognostic (all P>0.05). Multivariate survival analyses revealed that relative mean TBR changes were most potent in predicting response to regorafenib (P=0.038; HR, 0.246) and absolute mean TBR values for prognostication (P<0.001; HR, 0.005). CONCLUSION Data suggest that FET PET parameters are clinically valuable for identifying responders to regorafenib early after treatment initiation and helpful for prognostication. MRI parameters, including ADC values, do not appear to add valuable information in this context.

Characterization of MCL-1 in patients with colorectal cancer (CRC): Expression, molecular profiles, and outcomes.

3085 Background: Myeloid cell leukemia 1 (MCL-1) is a member of the BCL-2 protein family and is anti-apoptotic/pro-survival in function. Dysregulation of MCL-1 expression has been reported in several solid tumors, including lung and breast cancer. In CRC, MCL-1 has been associated with resistance to chemotherapeutic drugs and multi-kinase inhibitor regorafenib. Our study aimed to characterize the molecular features associated with MCL-1 gene expression in CRC. Methods: 28,576 CRC samples were analyzed by Caris Life Sciences (Phoenix, AZ) with WTS (Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES). MCL-1 expression was stratified by quartiles where top quartile transcripts per million (TPM) were considered high (Q4) and bottom quartile low (Q1). Cell infiltration (CI) in the tumor microenvironment (TME) was estimated by RNA deconvolution analysis using QuantiSEQ. Interferon-gamma and T-cell inflamed signatures were also calculated from RNA data. X2 and Fisher-Exact tests were used, and statistical significance was determined as a P-value adjusted for multiple comparisons ( q < 0.05). Real world survival was obtained from insurance claims data and Kaplan-Meier estimates were calculated for molecularly defined patients. Results: MCL-1 expression was significantly increased in rectal tumors compared to other primary tumor sites (median TPM [mTPM] 20.8 vs 18.9 vs 18.7, in rectal vs left-sided vs right-sided, respectively, q < 0.0001). MCL-1 high tumors were associated with increased rate of PD-L1 IHC levels (Q1 vs Q4: 4.2% vs 9.7% [all CRC]; 3.7% vs 8.9% [pMMR/MSS], q < 0.0001), while MCL-1-low showed marginally increased rates of TMB-high (8.0% vs 7.0% [all CRC]; 3.3% vs 2.6% [pMMR/MSS], P < 0.05). MCL-1-high expression was associated with a higher T-cell inflamed signature and IFN score ( q < 0.0001). MCL-1 high was associated with higher mutation rates of CDKN2A, BRCA2, KRAS and GNAS, while lower mutation rates of TP53, PIK3CA, PTEN, BRAF, APC, FBXW7, AMER1, SOX9, and SMAD2 and copy number amplifications in several genes ( q < 0.05). MCL-1 high TME had higher CI of M1 and M2 macrophages, monocytes, B cells, NK cells, and T-reg infiltration, while dendritic cells and CD4+ T-cell were lower ( q < 0.05). Patients with high MCL-1 expressing CRC had longer survival (Q4 vs Q1: 33.5 vs 24.8 months, HR 0.72, 95% CI 0.67-0.77, P < 0.0001) and longer time-on-treatment with regorafenib although with limited clinical benefit (Q4 vs Q1: 2.03 vs 1.06 months, HR: 0.67, 95% CI: 0.50-0.89, P = 0.005). Conclusions: Our data show a strong correlation between distinct immune biomarkers, TME cell infiltration and MCL-1 expression in CRC. Furthermore, increased tumor MCL-1 expression improved patient prognosis and treatment outcomes. These findings suggest a key clinical role for MCL-1 as an important modulator of anti-tumor immunity and TME and a potential biomarker in CRC.

Characterization of Glioblastoma Cells Response to Regorafenib.

Glioblastoma (GBM), the most malignant primary brain tumor in adults. Although not frequent, it has a relevant social impact because the peak incidence coincides with the age of professional maturity. A number of novel treatments have been proposed, yet clinical trials have been disappointing. Recently, a phase II clinical trial (REGOMA) demonstrated that the multikinase inhibitor regorafenib significantly increased the median overall survival (OS) of GBM patients when compared to lomustine-treated patients. On this basis, the National Comprehensive Cancer Network (NCCN) 2020 Guidelines included regorafenib as a preferred regimen in relapsed GBM treatment. Despite the use in GBM patients' therapy, little is known about the molecular mechanisms governing regorafenib effectiveness on the GBM tumor. Here we report an in vitro characterization of GBM tumor cells' response to regorafenib, performed both on cell lines and on patient-derived glioma stem cells (GSCs). Overall, regorafenib significantly reduced cell growth of 2D tumor cell cultures and of 3D tumor spheroids. Strikingly, this effect was accompanied by transcriptional regulation of epithelial to mesenchymal transition (EMT) genes and by an increased ability of surviving tumor cells to invade the surrounding matrix. Taken together, our data suggest that regorafenib limits cell growth, however, it might induce an invasive phenotype.

Repeated Contrast-Enhanced Micro-CT Examinations Decrease Animal Welfare and Influence Tumor Physiology.

Computed tomography (CT) imaging is considered relatively safe and is often used in preclinical research to study physiological processes. However, the sum of low-dose radiation, anesthesia, and animal handling might impact animal welfare and physiological parameters. This is particularly relevant for longitudinal studies with repeated CT examinations. Therefore, we investigated the influence of repeated native and contrast-enhanced (CE) CT on animal welfare and tumor physiology in regorafenib-treated and nontreated tumor-bearing mice. Mice bearing 4T1 breast cancer were divided into 5 groups: (1) no imaging, (2) isoflurane anesthesia only, (3) 4 mGy CT, (4) 50 mGy CT, and (5) CE-CT (iomeprol). In addition, half of each group was treated with the multikinase inhibitor regorafenib. Mice were imaged 3 times within 1 week under isoflurane anesthesia. Behavioral alterations were investigated by score sheet evaluation, rotarod test, heart rate measurements, and fecal corticosterone metabolite analysis. Tumor growth was measured daily with a caliper. Tumors were excised at the end of the experiment and histologically examined for blood vessel density, perfusion, and cell proliferation. According to the score sheet, animals showed a higher burden after anesthesia administration and in addition with CT imaging ( P < 0.001). Motor coordination was not affected by native CT, but significantly decreased after CE-CT in combination with the tumor therapy ( P < 0.001). Whereas tumor growth and blood vessel density were not influenced by anesthesia or imaging, CT-scanned animals had a higher tumor perfusion ( P < 0.001) and a lower tumor cell proliferation ( P < 0.001) for both radiation doses. The most significant difference was observed between the control and CE-CT groups. Repeated (CE-) CT imaging of anesthetized animals can lead to an impairment of animal motor coordination and, thus, welfare. Furthermore, these standard CT protocols seem to be capable of inducing alterations in tumor physiology when applied repetitively. These potential effects of native and CE-CT should be carefully considered in preclinical oncological research.

P11.53.B Efficacy and tolerability of regorafenib in pretreated patients with progressive CNS grade 3 or 4 gliomas

Abstract Background The phase 2 REGOMA trial suggested an encouraging overall survival benefit in glioblastoma patients at first relapse treated with the multikinase inhibitor regorafenib. Here, we evaluated the efficacy and side effects of regorafenib in a real-life setting. Material and Methods From 2018-2021, 30 patients with progressive WHO CNS grade 3 or 4 gliomas treated with regorafenib (160 mg/d; first 3 weeks of each 4-week cycle) with individual dose adjustment depending on toxicity were retrospectively identified. Side effects were evaluated according to the Common Terminology Criteria for Adverse Events (version 5.0). MRI was obtained at baseline and after every second cycle. Tumor progression was assessed according to RANO criteria. After regorafenib initiation, the median progression-free survival (PFS) and overall survival (OS) were calculated. Results The median number of treatment lines before regorafenib was 2 (range, 1-4). The majority of patients (73%) had two or more pretreatment lines. At first relapse, 27% of patients received regorafenib. A total of 94 regorafenib cycles were administered (median number of cycles, 2; range, 1-9 cycles). Grade 3 and 4 side effects were observed in 47% and 7% of patients, respectively, and were not significantly increased in patients with two or more pretreatments (P&amp;gt;0.05). The most frequent grade 3 or 4 side effects were laboratory abnormalities (62%). PFS was 2.6 months (range, 0.8-8.2 months), and the OS was 6.2 months (range, 0.9-24 months). Conclusion In patients with progressive WHO grade 3 or 4 gliomas, predominantly with two pretreatment lines or more, regorafenib seems to be effective despite considerable grade 3 or 4 side effects.

Studies on Biological and Molecular Effects of Small-Molecule Kinase Inhibitors on Human Glioblastoma Cells and Organotypic Brain Slices.

Glioblastoma is the most common and aggressive primary brain tumor. Multiple genetic and epigenetic alterations in several major signaling pathways—including the phosphoinositide 3-kinases (PI3K)/AKT/mTOR and the Raf/MEK/ERK pathway—could be found. We therefore aimed to investigate the biological and molecular effects of small-molecule kinase inhibitors that may interfere with those pathways. For this purpose, patient-derived glioblastoma cells were challenged with dactolisib, ipatasertib, MK-2206, regorafenib, or trametinib. To determine the effects of the small-molecule kinase inhibitors, assays of cell proliferation and apoptosis and immunoblot analyses were performed. To further investigate the effects of ipatasertib on organotypic brain slices harboring glioblastoma cells, the tumor growth was estimated. In addition, the network activity in brain slices was assessed by electrophysiological field potential recordings. Multi-kinase inhibitor regorafenib and both MK-2206 and dactolisib were very effective in all preclinical tumor models, while with respect to trametinib, two cell lines were found to be highly resistant. Only in HROG05 cells, ipatasertib showed anti-tumoral effects in vitro and in organotypic brain slices. Additionally, ipatasertib diminished synchronous network activity in organotypic brain slices. Overall, our data suggest that ipatasertib was only effective in selected tumor models, while especially regorafenib and MK-2206 presented a uniform response pattern.

Efficacy and tolerability of regorafenib in pretreated patients with progressive CNS grade 3 or 4 gliomas

BackgroundThe phase 2 REGOMA trial suggested an encouraging overall survival benefit in glioblastoma patients at first relapse treated with the multikinase inhibitor regorafenib. Here, we evaluated the efficacy and side effects of regorafenib in a real-life setting.MethodsFrom 2018 to 2021, 30 patients with progressive WHO CNS grade 3 or 4 gliomas treated with regorafenib (160 mg/day; first 3 weeks of each 4-week cycle) with individual dose adjustment depending on toxicity were retrospectively identified. Side effects were evaluated according to the Common Terminology Criteria for Adverse Events (version 5.0). MRI was obtained at baseline and after every second cycle. Tumor progression was assessed according to RANO criteria. After regorafenib initiation, the median PFS and OS were calculated.ResultsThe median number of treatment lines before regorafenib was 2 (range 1–4). Most patients (73%) had two or more pretreatment lines. At first relapse, 27% of patients received regorafenib. A total of 94 regorafenib cycles were administered (median 2 cycles; range 1–9 cycles). Grade 3 and 4 side effects were observed in 47% and 7% of patients, respectively, and were not significantly increased in patients with two or more pretreatments (P > 0.05). The most frequent grade 3 or 4 side effects were laboratory abnormalities (62%). PFS was 2.6 months (range 0.8–8.2 months), and the OS was 6.2 months (range 0.9–24 months).ConclusionsIn patients with progressive WHO grade 3 or 4 gliomas, predominantly with two pretreatment lines or more, regorafenib seems to be effective despite considerable grade 3 or 4 side effects.

A nonrandomized phase I and biomarker trial of regorafenib in advanced myeloid malignancies.

We conducted a single‐center, open‐label, dose escalation, and expansion phase I trial of the antiangiogenic multikinase inhibitor regorafenib in patients with advanced myeloid neoplasms. We enrolled 16 patients with relapsed/refractory acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), or myelodysplastic syndrome (MDS). A 3 + 3 dose escalation design was used with two planned dose levels (120 or 160 mg daily) and one de‐escalation level (80 mg daily). An additional 10 patients were treated on an expansion cohort. The recommended phase two dose of regorafenib was 160 mg daily, with no dose‐limiting toxicities. The best overall disease response by International Working Group criteria included one partial and stable disease in 11 patients. Tissue studies indicated no change in Ras/mitogen‐activated protein kinase (MAPK) pathway activation in responders. Pharmacodynamic changes in plasma VEGF, PlGF, and sVEGFR2 were detected during treatment. Baseline proinflammatory and angiogenic cytokine levels were not associated with clinical response. Single‐agent regorafenib demonstrated an acceptable safety profile in relapsed/refractory myeloid malignancy patients. Most patients achieved stable disease, with modest improvements in cell counts in some MDS patients. Biomarker studies were consistent with on‐target effects of regorafenib on angiogenesis. Future studies should investigate the role of regorafenib in combination therapy approaches.

Optimal solutions in the third line therapy for refractory metastatic colorectal cancer. CORRECTness and CONCURency

Colon cancer therapy currently includes at least 3 cytostatic agents and 6 targeted drugs, combinations of which constitute many different treatment regimens. Nevertheless, as shown by various clinical studies, the use of oxaliplatin, irinotecan and fluoropyrimidine regimens in conjunction with monoclonal targeted drugs remains the main one. After progression on the main lines of therapy and registration of refractory disease, there are not many standard options for treatment in the 3rd line that have statistical confidence in terms of improving survival rates. There have been attempts to search for genetic aberrations for targeted therapy, predictors of the effectiveness of immunotherapy, the use of re-introduction regimens (re-application of a regimen that the patient stopped taking due to intolerance phenomena) and re-challenge (use of previous regimens, with response at first time, and then developed progression), and the abundance of solutions could only be limited by the imagination of the researcher. However, with the advent of the new multi-kinase inhibitor regorafenib, recommendations for the treatment of colorectal cancer have changed. The drug has received indications for use in the third line of therapy for refractory colorectal cancer. A review of the existing options for targeted therapy and an analysis of studies of different regimens in the 3rd line of therapy in comparison with regorafenib was carried out. It has been established that regorafenib is currently the optimal option for the treatment of metastatic colorectal cancer in the third line of therapy, regardless of previous lines of therapy and mutational status, and has a satisfactory spectrum of side effects, many of which may be predictors of therapy efficacy.

Effect of Regorafenib on P2X7 Receptor Expression and Different Oncogenic Signaling Pathways in a Human Breast Cancer Cell Line: A Potential of New Insight of the Antitumor Effects of Regorafenib.

Background: Breast cancer is the most common malignancy in women worldwide. P2X7 is a transmembrane receptor expressed in breast cancer and activated by the ATP tumor microenvironment, driving cell proliferation, angiogenesis, and metastasis via different signaling pathways. The role of the P2X7 receptor, hypoxia, and autophagy in regulating tumor progression is controversial. The multikinase inhibitor regorafenib prevents the activation of numerous kinases involved in angiogenesis, proliferation, and metastasis. The present study aimed to evaluate the modulatory effect of regorafenib on the hypoxia/angiogenesis/P2X7R/autophagy axis on the MCF7 breast cancer cell line and its impact on different signaling pathways involved in breast cancer pathogenesis. Methods: The levels of VEGF, VEGFR, PI3K, NF-κB, HIF-1α, and LC3-II were analyzed using ELISA, and caspase-3 activity was also assessed colorimetrically. Phosphorylated (p)-p38 MAPK and purinergic ligand-gated ion channel 7 (P2X7) receptor protein expression levels were analyzed via Western blotting. Reverse transcription-quantitative PCR was used to determine the mRNA expression levels of Beclin 1 (BECN1), LC3-II, and sequestosome 1 (p62). Results: Regorafenib reduced MCF7 cell viability in a dose-dependent manner. Furthermore, regorafenib significantly reduced levels of PI3K, NF-κB, VEGF, VEGFR, P2X7 receptor, and p-p38 MAPK protein expression, and markedly reduced p62 mRNA expression levels. However, regorafenib significantly increased caspase-3 activity, as well as BECN1 and LC3-II mRNA expression levels. Conclusions: Regorafenib was demonstrated to possibly exhibit antitumor activity on the breast cancer cell line via modulation of the P2X7/HIF-1α/VEGF, P2X7/P38, P2X7/ERK/NF-κB, and P2X7/beclin 1 pathways.

NIMG-27. REGORAFENIB RESPONSE ASSESSMENT USING FET PET IN PATIENTS WITH PROGRESSIVE GLIOMA

Abstract BACKGROUND The REGOMA phase 2 trial showed an encouraging overall survival benefit of the multikinase inhibitor regorafenib in glioblastoma patients at first progression. We used O-(2-[18F]fluoroethyl)-L-tyrosine (FET) PET for the early assessment of response to regorafenib in patients with progressive glioma in an advanced disease stage. METHODS Thirty patients with progressive glioma were treated according to the REGOMA trial and prospectively followed. FET PET and MRI were performed at baseline and after the second cycle of regorafenib. Static PET parameters such as maximum and mean tumor-to-brain ratios (TBRmax, TBRmean) and metabolic tumor volumes (MTV) were calculated. Threshold values of FET PET parameters to predict a response were established by ROC analyses using an overall survival of ≥ 6 months as reference. The predictive value of FET PET parameters and their changes concerning overall survival was subsequently evaluated using the Kaplan-Meier test. MRI changes were evaluated according to the RANO criteria. RESULTS Up to now, 18 of 30 patients (glioblastoma, 83%; age range, 24-71 years) were eligible for data evaluation. The median number of tumor relapses before regorafenib was 2 (range, 1-4). During regorafenib (median cycles, 4; range, 2-9 cycles), CTCAE grade 3 or 4 side effects occurred in 56% and 11%, respectively. The median overall survival was 6 months (range, 3-18 months). After two cycles of regorafenib, a TBRmean reduction of 13% predicted a significantly longer overall survival (12 vs. 6 months; P=0.034). In contrast, MRI changes evaluated according to RANO criteria (i.e., Stable Disease or Partial Response vs. Progressive Disease) were not predictive (11 vs. 8 months; P=0.644). CONCLUSION Data suggest that amino acid PET using the tracer FET may be clinically valuable for identifying responders to regorafenib early after treatment initiation.

Triple MAPK Inhibition Salvaged a Relapsed Post BCMA CAR-T Cell Therapy in Multiple Myeloma Patient with BRAF V600E Dominant Clone

Background:Multiple Myeloma (MM) is a progressive plasma cell neoplasm characterized by heterogeneous clonal expansion. Despite numerous targeted therapy options, failing anti-BCMA CAR-T cell therapy represents a challenging treatment course with dismal prognosis. 53% of relapsed/refractory (rr) MM patients have shown emerging clones harboring mutations within the MAPK signaling pathway including the targetable BRAF V600E in 7% of cases. Allosteric kinase inhibition of MAPK pathway with BRAF monomer selective inhibitors +/- MEK inhibitor have been frequently attempted with rrMM patients with equivocal outcome on both disease progression and survival. This could be ascribed to the feedback activation of the pathway via BRAF dimers induction. In solid tumor contexts, a recent study has revealed that the multi-kinase inhibitor regorafenib is a potent and selective inhibitor of dimeric BRAF. Moreover, the study demonstrated overcoming of this negative feedback by combining two inhibitors targeting BRAF in its monomeric and its dimeric form, respectively, with MEK inhibition leading to more efficacious and tolerable treatment (Adamopoulos C. et al. Cancer Discov. 2021). Here, we show the applicability, effectiveness, and tolerability of the aforementioned triple therapy strategy in hematological malignancy context to salvage post BCMA CAR-T cell therapy in a BRAF V600E rrMM patient.Material and Methods:CD138 + selected cells were collected from bone marrow (BM) and patient's plasmacytoma before and after CAR-T cell therapy relapse, respectively. Samples were processed using the standard GATK 4 workflows and a consensus mutation calling approach was used to call somatic mutations using a matched normal obtained from peripheral blood. PhyloWGS (Deshwar et al, Genome Biol 2015) was then used to perform intra-tumor heterogeneity modeling and phylogeny estimation.CD138 + selected cells from BM of rrMM patient were treated with encorafenib (50nM) and binimetinib (250 nM) +/- regorafinib (1mM) for 48 hours, cells were harvested, lysed and immunoblotted with BRAF V600E and pERK antibodies.In parallel, rrMM patient BM selected CD138 + cells' sensitivity to MAPK inhibitors were assessed by Travera. Cells are incubated with different inhibitors at varying concentrations and combinations for 15 hours before having their single-cell mass distributions measured. Sensitivity is then characterized by the relative statistical difference (Hellinger distance) between conditions and untreated controls.A rrMM patient was started on targeted triple therapy using the following combination: BRAF monomer-inhibitor, dabrafenib (100mg, orally twice daily), a MEK-inhibitor, trametinib (1.5mg, orally for 21/28 days daily), and a BRAF dimer inhibitor, regorafenib (40mg, orally once daily).Results:Whole Exome Sequencing (WES) showed a BRAF V600E mutation was present prior to CAR-T cell therapy and persisted through subsequent treatments. The variant allele frequency of the mutation was 41% post CAR-T cell therapy (Figure 1A).Western blot of CD138 + cells confirmed BRAF V600E mutation with a specific antibody, and showed ERK phosphorylation, which was inhibited with trametinib. Longer exposure to trametinib confirmed a feedback loop activating MAPK which was abrogated with the BRAF dimer inhibitor regorafenib (Figure 1B).Travera analysis showed CD138 + cells' sensitivity to the combination of trametinib at 5mM, and dabrafenib at 1µM. Adding regorafenib at 1µM could slightly improve the response compared to the double combination. (Figure 1C).Clinically, three months of the triple therapy administration has led to prompt reduction in subcutaneous skin lesions as well as 80% reduction in free lambda chain (Figure 1D). Furthermore, the patient exhibited good tolerance to all three medications with minimal side effects (grade 1 fatigue). The treatment regimen allowed the patient to carry out activities of daily living and resume work.Conclusion:We have shown the applicability, effectiveness, and tolerability of the triple MAPK inhibition in a hematological malignancy context to salvage post BCMA CAR-T cell therapy in a rrMM patient with BRAF V600E mutation. Also, we have revealed the power of routine WES in tracking clonal evolution, and identifying targetable mutations which allows tailoring of therapeutics for MM patients. [Display omitted] DisclosuresStevens: Travera: Current Employment. Parekh: Foundation Medicine Inc: Consultancy; Amgen: Research Funding; PFIZER: Research Funding; CELGENE: Research Funding; Karyopharm Inv: Research Funding.

Long-term survival of a patient with microsatellite-stable refractory colorectal cancer with regorafenib and PD-1 inhibitor sintilimab: a case report and review of literature

BackgroundColorectal cancer (CRC) is the third most prevalent cancer worldwide and poses a serious challenge for clinicians. Previous studies have shown promising results in patients with Microsatellite Stable microsatellite-stable CRC refractory to chemotherapy upon treating with (Programmed Cell Death Protein 1) PD-1 inhibitor combined with regorafenib. Herein, we report a unique case of a patient for whom the conventional chemotherapy and radiotherapy were ineffective, but showed a prolonged stable disease with third-line treatment with regorafenib and PD-1 inhibitor, sintilimab.Case presentationA 64-year-old East Asian female patient was admitted to a regional cancer hospital presenting with abdominal unease due to increased stool frequency and bloody stool. Digital anal examination revealed adenocarcinoma, while genetic profiling of the tumor resections detected wild-type KRAS mutations in codon 12 and 13. Microsatellite instability (MSI) analysis for detecting germline mutations of (Mismatch-repair) MMR genes showed stable phenotype. In December 2016, Miles’ resection for intestinal adhesion release and iliac vessel exploration in the rectum was performed (Tumor, Node, Metastasis [TNM]: T3N0M0; stage IIA). The adjuvant chemotherapeutic regimen consisted of a combination of capecitabine at 1.5 g (twice daily) and oxaliplatin therapy at 200 mg for three cycles from February 2016; followed by administering capecitabine tablets orally (1.5 g bid) for five cycles as post-operative palliative care. The patient tested positive for hepatic C virus, which was managed by oral antiviral agents. Following recurrence of rectal adenocarcinoma after 4 years and disease progression with a previous chemotherapeutic regimen, regorafenib was administered at 120 mg once daily combined with sintilimab 200 mg, and the patient's progress was monitored. A follow-up computerized tomography imaging in March 2020 showed disease progression, additionally presented nodule formation (TNM: T3NxM1b; stage IVB). According to Response Evaluation Criteria in Solid Tumors criteria (RECIST), the patient showed a complete response (CR) after treatment with regorafenib and sintilimab immunotherapy.ConclusionData from this clinical case report support future exploration of combination treatment of the oral multi-kinase inhibitor regorafenib with PD-1 targeted monoclonal antibodies in patients with metastatic microsatellite-stable CRC.

P-090: BRAF V600E multiple myeloma patient salvaged with triple MAPK inhibition after CAR T relapse

<h3>Background</h3> Despite a growing arsenal of treatment choices, patient relapse post-BCMA-targeted CART therapy remains a challenge and the therapeutic path is still undefined. Among the most frequently observed actionable mutations, BRAF V600E, is present in ~7% of multiple myeloma (MM) patients. The availability of selective inhibitors of BRAF V600E makes this a valuable therapeutic target. However, the clinical efficacy of these inhibitors has been limited and short-lived, frequently due to feedback-induction of BRAF dimers. Novel studies revealed that the multi-kinase inhibitor regorafenib is a potent and selective inhibitor of dimeric BRAF. They have further demonstrated relief of these negative outcomes by inhibiting BRAF in both its monomeric and dimeric form in combination with MEK inhibition leading to more efficacious and tolerable treatment. (Adamopoulos C. et al. Cancer Discov. 21). We present a case of a MM patient with BRAF V600E mutation salvaged with a triple therapy inhibition strategy after relapsing following anti-BCMA CART therapy. <h3>Method</h3> A 61-year-old male with penta-refractory MM (IgA lambda), ISS stage 3 with hyperdiploidy, gain of 1q21 and del13 was treated with anti-BCMA CART therapy, achieving a best response of VGPR, and disease progression after 6 months. He was temporarily salvaged with BCNU/Mel ASCT and achieved a best response of PR until progression with extramedullary disease (subcutaneous skin lesions in lower extremities) and elevated lambda free light chains (FLC, 126.4mg/l) at 6 months. <h3>Results</h3> Targeted sequencing showed a BRAF V600E mutation was present prior to CART therapy in his bone marrow and persisted in his bone marrow and a plasmacytoma with a VAF of 41% at this current relapse. A western blot analysis of bone marrow aspirate confirmed BRAF V600E with a specific antibody, and showed phosphorylation of ERK, which was diminished with trametinib. Longer exposure to trametinib confirmed a feedback loop activating MAPK which was abrogated with the BRAF dimer inhibitor regorafenib. With these findings the patient was started on targeted therapy based on combination of a BRAF monomer-inhibitor, dabrafenib (100mg, orally twice daily), a MEK-inhibitor, trametinib (1.5mg, orally for 21/28 days daily), and a BRAF dimer inhibitor, regorafenib (40mg, orally once daily). Within 3 months of treatment initiation, prompt reduction in subcutaneous skin lesions and 80% reduction in free lambda FLC (27.5 mg/l) was observed. Furthermore, the patient had good tolerance to all three medications with minimal side effects (grade 1 fatigue) and continues treatment. The treatment regimen allowed the patient to carry out activities of daily living and return to work. <h3>Conclusion</h3> Post-CART failure treatment is a challenging and unmet need. NGS may identify targeted therapy that would be able to salvage patients with a tolerable side effect profile.

Regorafenib enhances anti-PD1 immunotherapy efficacy in murine colorectal cancers and their combination prevents tumor regrowth

BackgroundPatients with advanced colorectal cancer (CRC) have a poor prognosis. Combinations of immunotherapies and anti-angiogenic agents are currently being evaluated in clinical trials. In this study, the multikinase inhibitor regorafenib (REG) was combined with an anti-programmed cell death protein 1 (aPD1) antibody in syngeneic murine microsatellite-stable (MSS) CT26 and hypermutated MC38 colon cancer models to gain mechanistic insights into potential drug synergism.MethodsGrowth and progression of orthotopic CT26 and subcutaneous MC38 colon cancers were studied under treatment with varying doses of REG and aPD1 alone or in combination. Sustained effects were studied after treatment discontinuation. Changes in the tumor microenvironment were assessed by dynamic contrast-enhanced MRI, and histological and molecular analyses.ResultsIn both models, REG and aPD1 combination therapy significantly improved anti-tumor activity compared with single agents. However, in the CT26 model, the additive benefit of aPD1 only became apparent after treatment cessation. The combination treatment efficiently prevented tumor regrowth and completely suppressed liver metastasis, whereas the anti-tumorigenic effects of REG alone were abrogated soon after drug discontinuation. During treatment, REG significantly reduced the infiltration of immunosuppressive macrophages and regulatory T (Treg) cells into the tumor microenvironment. aPD1 significantly enhanced intratumoral IFNγ levels. The drugs synergized to induce sustained M1 polarization and durable reduction of Treg cells, which can explain the sustained tumor suppression.ConclusionsThis study highlights the synergistic immunomodulatory effects of REG and aPD1 combination therapy in mediating a sustained inhibition of colon cancer regrowth, strongly warranting clinical evaluation in CRC, including MSS tumors.