Combination Of Afatinib Research Articles

Patient-derived organoids and mini-PDX for predicting METN375S-mutated lung cancer patient clinical therapeutic response

Lung cancer as a molecularly and histologically high heterogonous disease, there is an urgent need to predict lung cancer patients' responses to anti-cancer treatment, and patient-derived organoids (PDOs) have been recognized as a valuable platform for preclinical drug screening. In this study, we successfully established 26 PDO lines from various subtypes of lung cancers including benign tumor, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, large-cell carcinoma, and small-cell carcinoma. These PDOs were shown to retain the major genomic and histological characteristics of primary tumors and remain stable during long-term culture. With the help of targeted genomic sequencing, we found that lung cancer that harbors METN375S mutation is selectively sensitive to afatinib, and a combination of afatinib and gemcitabine induced synthetic lethality in PDO and mini-PDX models. In summary, our findings demonstrate the potential of PDO in predicting lung cancer drug response, and reveal a promising strategy for METN375S mutant lung cancer treatment.

Afatinib plus PEM and CBDCA overcome osimertinib resistance in EGFR-mutated NSCLC with high thrombospondin-1 expression.

Osimertinib induces a marked response in non-small-cell lung cancer (NSCLC) patients harboring epidermal growth factor receptor (EGFR) gene mutations. However, acquired resistance to osimertinib remains an inevitable problem. In this study, we aimed to investigate osimertinib-resistant mechanisms and evaluate the combination therapy of afatinib and chemotherapy. We established osimertinib-resistant cell lines (PC-9-OR and H1975-OR) from EGFR-mutant lung adenocarcinoma cell lines PC-9 and H1975 by high exposure and stepwise method. Combination therapy of afatinib plus carboplatin (CBDCA) and pemetrexed (PEM) was effective in both parental and osimertinib-resistant cells. We found that expression of thrombospondin-1 (TSP-1) was upregulated in resistant cells using cDNA microarray analysis. We demonstrated that TSP-1 increases the expression of matrix metalloproteinases through integrin signaling and promotes tumor invasion in both PC-9-OR and H1975-OR, and that epithelial-to-mesenchymal transition (EMT) was involved in H1975-OR. Afatinib plus CBDCA and PEM reversed TSP-1-induced invasion ability and EMT changes in resistant cells. In PC-9-OR xenograft mouse models (five female Balb/c-Nude mice in each group), combination therapy strongly inhibited tumor growth compared with afatinib monotherapy (5 mg/kg, orally, five times per week) or CBDCA (75 mg/kg, intraperitoneally, one time per week) + PEM (100 mg/kg, intraperitoneally, one time per week) over a 28-day period. These results suggest that the combination of afatinib plus CBDCA and PEM, which effectively suppresses TSP-1 expression, may be a promising option in EGFR-mutated NSCLC patients after the acquisition of osimertinib resistance.

The Combination of Afatinib With Dasatinib or Miransertib Results in Synergistic Growth Inhibition of Stomach Cancer Cells.

Of various human epidermal growth factor receptor (HER) inhibitors, only the anti-HER2 monoclonal antibody (mAb) Herceptin/trastuzumab and the antibody-drug conjugate trastuzumab deruxtecan (T-Dxd) has been approved for the treatment of patients with stomach cancer. However, the duration of response may be short in many patients, with tumor heterogeneity being one contributing factor. We investigated the effect of various types of targeted agents on growth in vitro and migration of a panel of human stomach cancer cells (HSCCLs) and the impact of cell proliferation rate on the anti-tumor activities of these agents. We also investigated the association between the cell surface expression of the HER family members, hepatocyte growth factor receptor (c-Met), anaplastic lymphoma kinase (ALK)7 and cancer stem cell markers CD44 and CD133, and the response to the targeted agents. Of the 18 agents examined, the cyclin dependent kinase (CDK) 1/2/5/9 inhibitor dinaciclib was the most effective and inhibited the growth of all human HSCCLs at 50% inhibitory concentration (IC50) values between 9 nM to 23 nM. Of various HER inhibitors, the irreversible pan-HER family inhibitors (e.g., afatinib) were more effective than the reversible dual epidermal growth factor receptor (EGFR)/HER2 tyrosine kinase inhibitor (TKI) lapatinib and the EGFR-specific TKI erlotinib in inhibiting the growth of HSCCLs. Of agents targeting different downstream cell signaling molecules, dasatinib targeting Ab1/Src/C-Kit, trametinib targeting MERK1/2 and miransertib targeting AKT1/2/3 inhibited growth of majority of HSCCLs, with the IC50 values ranging from 2 nM to 7 µM. Many of these agents were more effective in inhibiting the growth of HSCCLs when they were proliferating at a slower rate. Treatment with neratinib, afatinib, dinaciclib, dasatinib, stattic, miransertib and paclitaxel significantly inhibited migration of stomach cancer cells. Interestingly, treatment with a combination of afatinib and dasatinib or afatinib and miransertib resulted in synergistic and additive growth inhibition of stomach cancer cells. These results suggest that treatment with a combination of these agents may be of therapeutic value in stomach cancer and warrants further investigations.

Gemcitabine and nab-paclitaxel combined with afatinib in metastatic pancreatic cancer – Results of a phase 1b clinical trial

PurposeThe combination of gemcitabine/nab-paclitaxel is an established standard treatment in the first-line treatment of metastatic ductal adenocarcinoma of the pancreas (mPDAC). Afatinib, an oral second-generation pan ErbB family tyrosine kinase inhibitor, has shown promising pre-clinical signs in the treatment of pancreatic cancer. The aim of this phase 1b trial was to determine the maximum tolerated dose (MTD) of afatinib in combination with gemcitabine/nab-paclitaxel in patients with mPDAC. MethodsTreatment naïve patients (≥18 years) with histologically proven mPDAC and good performance status (ECOG 0/1) were enrolled to receive gemcitabine/nab-paclitaxel in combination with afatinib. Treatment was continued until disease progression, or unacceptable toxicity. The primary endpoint MTD was determined using a 3 + 3 design. Treatment started at dose level 0 with intravenous gemcitabine/nab-paclitaxel 1000 mg/m2 / 125 mg/m2 (day 1, 8, 15 of a 28-day cycle) + oral afatinib 30 mg daily. At dose level + 1 afatinib was increased to 40 mg. Secondary endpoints included safety parameters and exploratory endpoints evaluated treatment efficacy. ResultsTwelve patients were included in this trial, and 11 patients were treated and analysed in the safety and full analysis set (FAS). At dose level 0 the first three patients did not experience a dose-limiting toxicity (DLT). At dose leve (DL) + 1 two patients experienced a DLT. Accordingly, enrolment continued at DL 0 with three more patients, of which one experienced DLT (skin rash ≥ CTCAE grade 3). Seven patients (63.6%) experienced at least one treatment-emergent serious adverse event (TESAE), with four patients (36.4%) experiencing TESAEs grade 3–5 related to the study medication. In the FAS, the objective response rate (ORR) was 36.4%, median progression-free survival (PFS) was 3.5 months and median overall survival in nine evaluable patients was 7.5 months. ConclusionsIn this phase 1b clinical trial, the MTD of gemcitabine/nab-paclitaxel (1000 mg/m2 / 125 mg/m2) and afatinib (30 mg) was established. In a cohort of 11 patients, the combination showed an acceptable safety profile.

Romidepsin and Afatinib Abrogate Jak–Signal Transducer and Activator of Transcription Signaling and Elicit Synergistic Antitumor Effects in Cutaneous T-Cell Lymphoma

Cutaneous T-cell lymphomas (CTCL) are mature lymphoid neoplasias resulting from the malignant transformation of skin-resident T cells. A distinctive clinical feature of CTCL is their sensitivity to treatment with histone deacetylase (HDAC) inhibitors. However, responses to HDAC inhibitor therapy are universally transient and non-curative, highlighting the need for effective and durable drug combinations. Here we demonstrate that the combination of romidepsin, a selective class I HDAC inhibitor, with afatinib, an epidermal growth factor receptor (EGFR) family inhibitor, induces strongly synergistic antitumor effects in CTCL models in vitro and in vivo via abrogation of JAK-STAT signalling. These results support a previously unrecognized potential role for HDAC inhibitor plus afatinib combination in the treatment of CTCL.

Curcumin potentiates the ErbB receptors inhibitor Afatinib for enhanced antitumor activity in malignant mesothelioma

Several attempts have been made to develop targeted therapies for malignant mesothelioma (MM), an aggressive tumour with a poor prognosis. In this study we evaluated whether Curcumin (CUR) potentiated the antitumor activity of the ErbB receptors inhibitor Afatinib (AFA) on MM, employing cell lines cultured in vitro and mice bearing intraperitoneally transplanted, syngeneic MM cells. The rationale behind this hypothesis was that CUR could counteract mechanisms of acquired resistance to AFA. We analysed CUR and AFA effects on MM cell growth, cell cycle, autophagy, and on the modulation of tumour-supporting signalling pathways. This study demonstrated that, as compared to the individual compounds, the combination of AFA + CUR had a stronger effect on MM progression which can be ascribed either to increased tumour cell growth inhibition or to an enhanced pro-apoptotic effect. These results warrant future studies aimed at further exploring the therapeutic potential of AFA + CUR-based combination regimens for MM treatment.

A phase II study of afatinib in combination with pemetrexed and carboplatin in patients with EGFR mutation positive non-squamous, advanced non–small-cell lung cancer (NSCLC) refractory to first-line osimertinib treatment: NEJ025B.

9044 Background: To date, osimertinib has been commonly used as a first-line treatment for EGFR mutated advanced NSCLC. However, the issue of what constitutes effective treatment after osimertinib failure remains．We evaluated treatment with afatinib plus chemotherapy for EGFR mutated NSCLC resistant to osimertinib. Methods: Patients (pts) with EGFRm (Del19 or L858R) after failure of osimertinib treatment were assigned to a regimen of afatinib 20mg daily combined with carboplatin AUC5 mg/ml/min and pemetrexed 500mg/m2 every 3 weeks followed by maintenance treatment with afatinib plus pemetrexed until disease progression or unacceptable toxicity was noted. The primary endpoint was rate of PFS at 6 months after initiation of treatment (6M-PFSR). The threshold was set at 35% and the expected value at 60% (two-sided P-value of 5% and power of 80%). Thirty-one patients were required, and the target number of patients was set at 35 after accounting for ineligible cases. Secondary endpoints were PFS, OS, ORR, DOR, and Safety. In this study, blood samples were collected before and after study treatment and at the time of PD to examine biomarkers using CAPP-SEQ. This biomarker study is ongoing. Results: Between June 7, 2020 and January 19, 2022, 36 pts were enrolled. One patient was found to meet the exclusion criteria, and the efficacy was analyzed in the other 35 patients. The mean age was 70 years, 60% were women, and 54.3% were nonsmokers. The 6M-PFSR, the primary endpoint, was 57.1%. The confidence interval of 39.3%-71.5% exceeded the threshold of 35%, and the study met its primary endpoint. Notably, 24.1% of pts had a long-term PFS of 1 year or longer. ORR was 48.6%, DCR was 88.6%, median PFS was 8.2M, median DOR was 5.6 M, and median OS was not reached. By mutation type, ORR were similar for Del19 and L858R (46.7% and 50.0%, respectively), but median PFS tended to be longer for Del19 compared to L858R (9.6M and 5.2M, respectively). Pts who responded to previous osimertinib therapy (CR and PR: n = 29) tended to have longer mPFS compared to non-responders (SD, PD, and NE: n = 6) (8.5M and 5.8M, respectively). Adverse events due to TKI and chemotherapy were observed. Diarrhea (52.8%), anorexia (44.4%), fatigue (33.3%), and paronychia (36.1%) were the most frequent adverse events, and these adverse events were predictable and manageable. Interstitial pneumonia developed in three patients (8%), one of whom was the only death in the study. Conclusions: The combination of afatinib and the platinum doublet showed satisfactory efficacy with manageable adverse events in tumors refractory to osimertinib, and met its primary endpoint. OS follow-up and biomarker analyses are still ongoing. This regimen is expected to be a candidate for second-line therapy after osimertinib. Clinical trial information: jRCTs021200005 .

Afatinib combined with anti-PD1 enhances immunotherapy of hepatocellular carcinoma via ERBB2/STAT3/PD-L1 signaling.

Afatinib is mainly used to treat advanced non-small cell lung cancer, but its therapeutic effect on hepatocellular carcinoma is still unclear. Over 800 drugs were screened by CCK8 technology and afatinib was found to have a significant inhibitory effect on liver cancer cells. The expression of PDL1 in tumor cells treated with drugs were detected by qRT-PCR and Weston Blot experiments. The effects of afatinib on the growth, migration and invasion of HCC cells were evaluated using wound healing, Transwell, and cell cloning assays. The in vivo effects of afatinib in combination with anti-PD1 were evaluated in C57/BL6J mice with subcutaneous tumorigenesis. Bioinformatics analysis was performed to explore the specific mechanism of afatinib's inhibition of ERBB2 in improving the expression level of PD-L1, which was subsequently verified through experiments. Afatinib was found to have a significant inhibitory effect on liver cancer cells, as confirmed by in vitro experiments, which demonstrated that it could significantly suppress the growth, invasion and migration of HCC cells. qRT PCR and Weston Blot experiments also showed that Afatinib can enhance the expression of PD-L1 in tumor cells. In addition, in vitro experiments confirmed that afatinib can significantly enhance the immunotherapeutic effect of hepatocellular carcinoma. Afatinib's ability to increase PD-L1 expression is mediated by STAT3 activation following its action on HCC cells. Afatinib enhances PD-L1 expression in tumor cells through the STAT3/PD-L1 pathway. The combination of afatinib and anti-PD1 treatment significantly increases the immunotherapeutic effect of HCC.

A real-world study of Afatinib plus ramucirumab in treatment-naïve, EGFR-mutated, non–small cell lung cancer

BackgroundRecent reports suggested combining ramucirumab with epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) to overcome EGFR resistance in non–small cell lung cancer (NSCLC). Nonetheless, evidence supporting the activity of afatinib and ramucirumab is lacking. This study investigated the survival benefits and safety profile of afatinib plus ramucirumab in patients with treatment-naïve, EGFR-mutated, metastatic NSCLC.Materials and methodsThe medical records of patients with EGFR-mutated NSCLC were retrospectively retrieved. Patients who received first-line sequential afatinib followed by ramucirumab and the first-line combination of afatinib plus ramucirumab were included. The Kaplan-Meier was used to estimate the progression-free survival (PFS) of all included patients, patients on sequential afatinib followed by ramucirumab (PFS1), and patients on the up-front combination of afatinib and ramucirumab (PFS2).ResultsThirty-three patients were included (25 women; median age: 63 [45–82] years). The median follow-up of the included patients was 17 months (range 6–89 months). the median PFS for the whole cohort was 71 months (95% CI 67.2–74.8) with eight events during the follow-up. The median PFS1 and PFS2 were 71 months (95 CI not defined) and 26 months (95% CI 18.6–33.4), respectively. In terms of OS, the median OS for all patients and patients on sequential treatment was not defined, while the median OS for patients on upfront combination was 30 months (95% CI 20.9–39.1). There was no significant association between EGFR mutation type and PFS1 or PFS2.ConclusionsAfatinib plus ramucirumab could improve the PFS of patients with EGFR-positive NSCLC at a predictable safety profile. Our data also suggest a survival benefit of adding ramucirumab to afatinib in patients with uncommon mutations, which should be investigated further.

Abstract B015: Wildtype RAS activity and PI3K signaling as new vulnerabilities in cells with acquired resistance to sotorasib

Abstract Purpose: Sotorasib (AMG510) has demonstrated remarkable response in lung cancer patients with tumors driven by oncogenic KRASG12C mutation. However, recently published clinical data identified acquired mutations in RAS and other genomic alterations as potential mechanisms of resistance. The cause of resistance in more than half of the patient cohort was undetermined with genomic sequencing. We hypothesize that rewiring of signaling networks could be the newly acquired vulnerabilities in cells progressing after sotorasib treatment. Experimental Design: We performed whole exome sequencing, transcriptomic profiling, and mass spectrometry-based proteomics/phosphoproteomics of the parental and sotorasib resistant isogenic line of LU65 (LU65-AMGR). The omics analyses were integrated with functional screens to prioritize a set of druggable targets/pathways. Results: We did not observe obvious acquired mutations that drive resistance to sotorasib in LU65-AMGR cells. To investigate compensatory signaling critically important for the survival/growth of LU65-AMGR cells, we studied signaling perturbations using mass spectrometry-based phosphoproteomics approach. We identified 430 and 1,574 phosphosites differentially expressed in resistant cells (± 1.5-fold & p < 0.05) with phosphotyrosine (pY) and global phosphoproteome (pS/T/Y) enrichment, respectively. Analysis of phosphoproteomics data identified increased phosphorylation of multiple RTKs including EGFR, HER2, HER3, IGF1R, AXL and PDGFRA, including others. RAS-GTP pull-down show increased levels of RAS-GTP and NRAS-GTP in the resistant cells. RAS isoform quantification using parallel reaction monitoring (MRM) mass spectrometry further confirmed increased activity of NRAS in pull-down samples. Transcriptomic analysis revealed elevated genes responsible for anti-apoptosis pathways mediated by PI3K/AKT signaling. Enrichment of transcription factors such as AP1 and AP-2A could drive enhanced EGF expression and EGFR phosphorylation in sotorasib resistant cells. Consistent with enhanced RTK and RAS activity, Western blots confirmed higher phosphorylation of ERK and AKT in LU65-AMGR cells. We show higher EGFR phosphorylation in resistant cells when compared to the parental counterpart. While LU65 cells viability is reduced markedly by sotorasib combination with afatinib, an irreversible HER family inhibitor, resistant cells showed lesser effect on cell viability with afatinib combination. Consistent with our in vitro observations, the sotorasib and afatinib combination treatment significantly regressed tumor growth only in LU65 xenografts, but not in LU65-AMGR xenografts. Conclusions: Our data suggest that activation of multiple RTKs maintains RAS activity and PI3K signaling in LU65-AMGR cells. Thus, dual pan-RAS and PI3K inhibition could serve as promising strategy of treatment in relapsed tumors identified with WT RAS and PI3K signaling activation. Citation Format: Denis Imbody, Hitendra S. Solanki, Bina Desai, Paul A. Stewart, Yaakov Stern, Ryoji Kato, Anurima Majumder, Liznair Bridenstine, Aobuli Xieraili, Bin Fang, Lancia Darville, Fumi Kinose, John M. Koomen, Uwe Rix, Andriy Marusyk, Eric B. Haura. Wildtype RAS activity and PI3K signaling as new vulnerabilities in cells with acquired resistance to sotorasib [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B015.

Abstract 6132: Impact of treatment with agents targeting different members of HER family, CDKs and downstream cell signaling molecules on growth and migration of stomach cancer cells

Abstract Stomach cancer ranks fifth and fourth for the incidence and mortality rate for cancer worldwide, respectively. In recent years, several inhibitors with specificity to one or more members of human epidermal growth factor receptor (HER) family have been approved for the treatment of patients with different cancer types. Of these, only the anti-HER2 monoclonal antibody (mAb) Herceptin/trastuzumab and the antibody-drug conjugate trastuzumab deruxtecan has been approved for the treatment of patients with stomach cancer. However, the duration of response may be short in many patients and with tumor heterogeneity being one contributing factor. In this study, we investigated the effect of various types of targeted agents on the growth in vitro of a panel of human stomach cancer cells (HSCCLs) and the impact of stomach cancer proliferation rate on the anti-tumor activities of these agents. Moreover, we investigated the cell surface expression of the HER family members, other biomarkers such as C-Met, Alk-7 and cancer stem cell makers CD44 and CD133 by flow cytometry and the effect various targeted agents on tumor migration using Incucyte. Of the 18 agents examined, the CDK 1/2/5/9 inhibitor dinaciclib was the most effective and inhibited the growth of all human HSCCLs at IC50 values between 1nM to 9nM. Of various HER inhibitors, the irreversible pan-HER family inhibitors (e.g., afatinib) were more effective than the reversible dual EGFR/HER2 TKI lapatinib and the EGFR specific TKI erlotinib in inhibiting the growth of HSCCLs. Interestingly, the HER-2 overexpressing cells lines NCI-N87 (Mean Fluorescence Intensity =596) was most sensitive to the HER inhibitors. Of agents targeting different downstream cell signaling molecules, dasatinib targeting Ab1/Src/C-Kit, trametinib targeting MERK1/2 and miransertib targeting AKT1/2/3 inhibited growth of majority of HSCCLs and with the IC50 values ranging from 2nM to 7uM. Interestingly, many of these agents were more effective in inhibiting the growth of HSCCLs when proliferating at slower rate. Of the agent examined, neratinib, afatinib, dinacicilib, dasatinib, stattic and miransertib also inhibited the migration of stomach cancer cells. Finally, treatment with a combination of afatinib with dinaciclib, capmatinib, dasatinib, stattic, ponatinib or miransertib resulted in synergistic and additive growth inhibition of stomach cancer cells. As stomach cancer cells are heterogenous in nature, our results support further research on the therapeutic potential of the CDK dinaciclib in combination with a pan-HER inhibitor in stomach cancer. Citation Format: Tina Al-Janaby, Narmin Nahi, Said A. Khelwatty, Alan Seddon, Izhar Bagwan, Helmout Modjtahedi. Impact of treatment with agents targeting different members of HER family, CDKs and downstream cell signaling molecules on growth and migration of stomach cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6132.

Abstract 3999: Combination of T-DXd with the irreversible pan-HER TKI afatinib drives combination benefit in HER2-low gastric and lung tumors

Abstract Background: Trastuzumab deruxtecan (T-DXd) has been approved for advanced or metastatic breast, gastric and lung cancers. It has been postulated that irreversible tyrosine kinase inhibitors (TKIs) increase HER2 internalization, thus enhancing T-DXd efficacy. Here we report studies evaluating the combination of TKIs with T-DXd in preclinical models of HER2-low disease. Methods: Surface HER2 was measured with flow cytometry quantifying fluorescently labelled trastuzumab on the surface of HER2-low cell lines HCC38 and HCC1171 treated with afatinib, neratinib, lapatinib or tucatinib for 12 h. HER2 receptor dynamics on cell surface was also measured in NCI-N87 (HER2 IHC 3+) and HCC38 at 24, 48, 72 and 96 h post-treatment with afatinib, and recovery was measured up to 72 h after drug washout. In vivo efficacy was established with T-DXd alone and in combination with afatinib or tucatinib in two gastric patient-derived xenograft (PDX) models, HD-2 (HER2 IHC 1+) and FU (HER2 IHC 2+), and one lung PDX model, MEDI-NSCLC-04 (HER2 IHC 1+). Results: In both HER2-low cell lines, treatment with irreversible inhibitors neratinib or afatinib led to enhanced internalization with a greater than 50% surface HER2 reduction, while treatment with the reversible inhibitors lapatinib or tucatinib led to 13-35% higher surface HER2 levels. Washout experiments indicated that receptor recovery at the plasma membrane begins between 4-12h post-washout of afatinib, with complete recovery at about 72h. Tumor shrinkage was significant in HER2-low gastric PDXs when T-DXd was combined with afatinib, but not with tucatinib. In HD-2, T-DXd 10mg/kg with afatinib 20mg/kg, and T-DXd 3mg/kg with tucatinib 25mg/kg combinations resulted in 74% and -3% (with respect to T-DXd) tumor growth inhibition (TGI), respectively. In FU, T-DXd 3mg/kg with afatinib 20mg/kg, and T-DXd 3mg/kg with tucatinib 25mg/kg combinations resulted in 80.3% and 24.7% TGI, respectively. Similarly, in MEDI-NSCLC-04 PDX model, benefit was observed in 5mg/kg T-DXd combination with 20mg/kg QD afatinib (95.3% TGI with respect to monotherapy T-DXd), but not with tucatinib (0.37% TGI). Intermittent schedules (QW, MWF) were also tested for afatinib in combination with T-DXd, with the MWF showing 49.2% TGI and QW showing 33.0 %TGI. In tissue samples from MEDI-NSCLC-04 efficacy study, γH2AX was detected by IHC in T-DXd and T-DXd+afatinib combination. In western blots, a decrease in HER2 as well as pHER2 was observed in response to T-DXd and 20mg/kg afatinib treatment, but not with tucatinib. Conclusions: In HER2 low gastric and lung preclinical models, T-DXd combined with irreversible pan-HER inhibitor induced superior antitumor effects compared to single agents or the combination of T-DXd with reversible inhibitors. These data support the rationale to test combination of T-DXd and pan-HER irreversible inhibitors in the clinical setting. Citation Format: Deepa Bhavsar, Laura Kazlauskas, Flavia Michelini, Matt Griffin, Matt Wilson, Fabiola Cecchi, Liz Croydon, Kim Maratea, Elisa de Stanchina, Yelena Y. Janjigian, Maurizio Scaltriti, Theresa Proia, Jerome Mettetal. Combination of T-DXd with the irreversible pan-HER TKI afatinib drives combination benefit in HER2-low gastric and lung tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3999.

PD25-06 COMBINATION TREATMENT WITH INTRAVESICAL INTERFERON GENE THERAPY AND PAN-ERBB RECEPTOR FAMILY BLOCKER IMPROVES SURVIVAL IN MICE WITH BLADDER CANCER

You have accessJournal of UrologyCME1 Apr 2023PD25-06 COMBINATION TREATMENT WITH INTRAVESICAL INTERFERON GENE THERAPY AND PAN-ERBB RECEPTOR FAMILY BLOCKER IMPROVES SURVIVAL IN MICE WITH BLADDER CANCER Akshay Sood, Jan Rudzinski, Alberto Martini, Come Tholomier, Sharada Mokkapati, and Colin Dinney Akshay SoodAkshay Sood More articles by this author , Jan RudzinskiJan Rudzinski More articles by this author , Alberto MartiniAlberto Martini More articles by this author , Come TholomierCome Tholomier More articles by this author , Sharada MokkapatiSharada Mokkapati More articles by this author , and Colin DinneyColin Dinney More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003303.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Recently, intravesical interferon (IFNα) gene therapy has demonstrated success in treatment of BCG-unresponsive NMIBC in a large, randomized, phase-III trial. While the results have been promising, there is room for improvement. One strategy to improve treatment efficacy and durability may be to identify actionable resistance mechanisms and employ targeted combination therapies. METHODS: We performed end-tumor RNA-seq analysis of MB49 mice tumors treated with IFNα gene therapy to identify potential mechanisms of therapy resistance – ErbB pathway was discovered as a potential target (Figure 1a). We therefore tested combination treatment with ErbB pathway blocker and IFNα in MB49 cells using lentiviral IFNα (LV-IFNα) with/without Afatinib in vitro. Next, in vivo studies were conducted in the MB49 orthotopic mice bladder cancer model: mice were randomized in to 5 treatment groups (n=10 each), saline (Ctrl), LV-Ctrl, Afatinib monotherapy, LV-IFNα monotherapy, and the experimental LV-IFNα + Afatinib combination therapy. Overall survival was assessed. RESULTS: Combination therapy significantly reduced the viability of MB49 cells in vitro compared to any of the other treatment conditions (mean relative ATPase activity for combination treatment at 72 h 4%, compared to 100%, 26%, and 28% for Ctrl, LV-IFNα, and Afatinib, p<0.001; Figure 1b). This effect on cell viability appeared to be driven by a combination of additive cytostatic and cytolytic effects (Figure 1c-d). The combination treatment also decreased cell migration (mean migrated cells/10x field on Boyden chamber assay: 92.3 for combination therapy and 631.0, 600.4, and 270.3 for Ctrl, LV-IFNα, and Afatinib, p<0.001; Figure 1e). Finally, in vivo studies demonstrated improved OS with combination therapy (median OS 49 d in the combination group vs 15, 29, and 26 d in the Ctrl, LV-IFNα, and Afatinib groups, Log-rank p<0.001; Figure 1f). No mice in the combination group died of drug toxicity. CONCLUSIONS: Our preliminary investigations suggest that ErbB pathway may be a clinically meaningful resistance mechanism, which when targeted alongside IFNα gene therapy may improve overall treatment efficacy. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e731 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Akshay Sood More articles by this author Jan Rudzinski More articles by this author Alberto Martini More articles by this author Come Tholomier More articles by this author Sharada Mokkapati More articles by this author Colin Dinney More articles by this author Expand All Advertisement PDF downloadLoading ...

Bruceine D and afatinib combination inhibits ovarian cancer cells proliferation and migration through DNA damage repair and EGFR pathway.

Owing to the high rates of relapse and migration, ovarian cancer (OC) has been recognized as the most lethal gynecological malignancy worldwide. The activity of the epidermal growth factor receptor (EGFR) signaling pathway is frequently associated with OC cell proliferation and migration. Despite this knowledge, inhibition of EGFR signaling in OC patients failed to achieve satisfactory therapeutic effects. In this study, we identified that bruceine D (BD) and EGFR inhibitor, afatinib, combination resulted in synergistic anti-OC effects. The results indicated that compared with one of both drugs alone, the combination of BD and afatinib slowed the DNA replication rate, inhibition of cell viability, and proliferation and clone formation. This resulted in cell cycle arrest and cell apoptosis. In addition, the combination of BD and afatinib possessed a stronger ability to inhibit the OC cell adhesion and migration than treatment with BD or afatinib alone. Mechanistically, the combined treatment triggered intense DNA damage, suppressed DNA damage repair, and enhanced the inhibition of the EGFR pathway. These results demonstrated that compared with each pathway inhibition, combined blocking of both DNA damage repair and the EGFR pathway appears to more effective against OC treatment. The results support the potential of BD and afatinib combination as a therapeutic strategy for OC patients.

Afatinib in combination with GEMOX chemotherapy as the adjuvant treatment in patients with ErbB pathway mutated, resectable gallbladder cancer: study protocol for a ctDNA-based, multicentre, open-label, randomised, controlled, phase II trial

IntroductionGallbladder cancer (GBC) is an aggressive type of digestive system cancer with a dismal outcome. Given the lack of effective treatment options, the disease rapidly reoccurs and 5-year survival rate...

Afatinib plus osimertinib in the treatment of osimertinib-resistant non-small cell lung carcinoma: a phase I clinical trial

BackgroundConquering acquired resistance to osimertinib remains a major challenge in treating patients with epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer (NSCLC). Thus, we aimed to determine the safety and efficacy of combination treatment with osimertinib and afatinib for patients with acquired resistance to osimertinib.MethodsThis open-label phase I study was a feasibility study of the combination of afatinib and osimertinib for patients with advanced EGFR-positive NSCLC who had progressive disease after receiving osimertinib. The primary endpoint was to determine the maximum tolerated dose (MTD). We enrolled patients who received afatinib at three different dose levels (level 1, 20 mg; level 2, 30 mg; level 3, 40 mg) combined with osimertinib at a standard dose of 80 mg once per day.ResultsThirteen patients were enrolled in this study. The MTD was defined as 30 mg afatinib when combined with daily oral administration of osimertinib (80 mg). The most frequent adverse events were diarrhea (76.9%), anemia (76.9%), and rash (69.2%). Considering the toxicity profiles during all treatment periods, the recommended oral dose of afatinib was determined as 20 mg daily, with an osimertinib dose of 80 mg. For all evaluable patients (n = 12), the response rate was 7.7% and the disease-control rate was 46.2%.ConclusionCombination therapy with osimertinib and afatinib was tolerable; however, the synergistic effect of afatinib with osimertinib may be limited in osimertinib-resistant patients.Trial registrationJapan Registry of Clinical Trials ID: jRCTs051180008, registered date: 08/11/2018.

Afatinib Reverses EMT via Inhibiting CD44-Stat3 Axis to Promote Radiosensitivity in Nasopharyngeal Carcinoma

Afatinib, a second-generation tyrosine kinase inhibitor (TKI), exerts its radiosensitive effects in nasopharyngeal carcinoma (NPC). However, the detailed mechanism of afatinib-mediated sensitivity to radiation is still obscure in NPC. Quantitative phosphorylated proteomics and bioinformatics analysis were performed to illustrate the global phosphoprotein changes. The activity of the CD44-Stat3 axis and Epithelial-Mesenchymal Transition (EMT)-linked markers were evaluated by Western blotting. Wound healing and transwell assays were used to determine the levels of cell migration upon afatinib combined IR treatment. Cell proliferation was tested by CCK-8 assay. A pharmacological agonist by IL-6 was applied to activate Stat3. The xenograft mouse model was treated with afatinib, radiation or a combination of afatinib and radiation to detect the radiosensitivity of afatinib in vivo. In the present study, we discovered that afatinib triggered global protein phosphorylation alterations in NPC cells. Further, bioinformatics analysis indicated that afatinib inhibited the CD44-Stat3 signaling and subsequent EMT process. Moreover, functional assays demonstrated that afatinib combined radiation treatment remarkably impeded cell viability, migration, EMT process and CD44-Stat3 activity in vitro and in vivo. In addition, pharmacological stimulation of Stat3 rescued radiosensitivity and biological functions induced by afatinib in NPC cells. This suggested that afatinib reversed the EMT process by blocking the activity of the CD44-Stat3 axis. Collectively, this work identifies the molecular mechanism of afatinib as a radiation sensitizer, thus providing a potentially useful combination treatment and drug target for NPC radiosensitization. Our findings describe a new function of afatinib in radiosensitivity and cancer treatment.

Neoadjuvant afatinib with paclitaxel for triple-negative breast cancer and the molecular characteristics in responders and non-responders

The prognosis of triple-negative breast cancer (TNBC) is worse and a major proportion of TNBC expresses epidermal growth factor receptor (EGFR). Afatinib can inhibit EGFR signal pathway; however, its treatment effect for TNBC is unknown. Thus, we aimed to assess the efficacy and biomarkers of afatinib in combination with paclitaxel in a neoadjuvant setting. Patients with stage II to III TNBC were enrolled. They received 40mg of afatinib daily for 14 days, followed by daily afatinib and weekly paclitaxel (80mg/m2) every 21 days for four to six cycles. To explore the mechanisms of responsiveness and non-responsiveness, 409 cancer-associated genes were sequenced. Twenty-one patients were enrolled and one patient achieved a complete clinical response; however, a 2mm residual tumor was noted in the surgical specimen. Overall, 33.0% patients were responders. Fifteen patients received molecular testing. No activated mutation of EGFR or Her2 were found. Activated PI3K or JAK2 pathway were trended to associate with non-responder (p=0.057). Mutation of homologous recombination (HR) genes were correlated with non-responsiveness (p=0.005). Seven patients did not have altered PI3K, JAK2 or HR pathway; six (85.7%) of them were responder. Patients with the amplified DAXX gene was associated with a favorable trend of response (p=0.109). Adding afatinib to neoadjuvant paclitaxel generated a modest effect in TNBC. Exploratory molecular analysis suggested that activated PI3K, JAK2 pathways and mutation of HR genes were associated with therapeutic non-responsiveness, and amplification of DAXX genes was associated with responsiveness to afatinib in combination with paclitaxel.

Abstract 5153: Longitudinal cell-free DNA analysis in phase I study evaluating afatinib in combination with osimertinib in patients who failed prior osimertinib treatment

Abstract Background: To overcome resistance to EGFR TKI osimertinib in EGFR-mutated lung cancer is critically needed. We performed a biomarker study utilizing serially collected plasma samples in a phase I study to investigate the safety and efficacy of the combination of osimertinib and afatinib in patients who failed osimertinib (jRCTs051180008), in which a total of 13 patients were enrolled and the overall response rate and the median progression-free survival were 7.7% and 2.4 months, respectively, to access the trajectory of the mutational landscape and its association with the efficacy. Methods: Peripheral blood samples were collected from the patients in K2 EDTA vacutainers (Becton Dickinson) before and four weeks after administration of the combination therapy and upon disease progression. Plasma was isolated immediately after the blood draw and stored at -80℃ until use. Cell-free DNA (cfDNA) was extracted from the plasma and used for next-generation sequencing library construction using AVENIO ctDNA Surveillance Kit (Roche Diagnostics) to assess genetic alterations in 197 cancer-related genes. Sequencing was performed on Nextseq500 system (Illumina) followed by mutation analyses using an Avenio Oncology Analysis Server (Roche Diagnostics). Results: Mutations in EGFR gene were detected in 10 out of 13 cfDNA samples (77%) including T790M/cis-C797S mutations before treatment and among those harboring T790M/cis-C797S mutations, 2 patients had progressive disease (PD) and 1 had stable disease (SD), indicating little benefit from the combination therapy in this genotype. Although other mutations in genes such as TP53 and/or KRAS were detected before treatment, no relevant associations were observed between tumor response or progression-free survival. One patient who had SD harbored CNTN5 mutation at PD, suggesting the potential involvement with the acquired resistance. Early emergence of EGFR G796S mutation observed in one patient after the initiation of the treatment along with basal L858R/T790M mutations may confer early resistance to the treatment. We also observed the emergence of MET amplification in 2 patients and ERBB2 amplification in 1 patient at PD, strongly suggesting the potential mechanisms responsible for resistance. Conclusions: This signal seeking biomarker study using cfDNA to better understand the association of mutational status and the treatment efficacy of the combined treatment was feasible and informative despite the small-scale study, supporting the advantage of further implementation. Citation Format: Yasuhiro Koh, Satoru Miura, Jun Oyanagi, Hiroshige Yoshioka, Koichi Azuma, Hidenobu Ishii, Kayoko Kibata, Kenichi Koyama, Shunsuke Teraoka, Yuichi Ozawa, Takaaki Tokito, Toshio Shimokawa, Takayasu Kurata, Nobuyuki Yamamoto, Hiroshi Tanaka. Longitudinal cell-free DNA analysis in phase I study evaluating afatinib in combination with osimertinib in patients who failed prior osimertinib treatment [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 5153.

Simultaneously targeting ErbB family kinases and PI3K in HPV-positive head and neck squamous cell carcinoma

ObjectivesTo identify the most effective PI3K and EGFR inhibitors in HPV-positive head and neck squamous cell carcinoma (HNSCC) and investigate the efficacy of a combination of an ErbB family kinase inhibitor and a PI3K inhibitor to inhibit cell proliferation of HPV-positive HNSCC. Materials and methodHPV-positive HNSCC cell lines were treated with the FDA approved ErbB kinase inhibitor, Afatinib or FDA-approved PI3K inhibitor, Copanlisib, alone or in combination, and phosphorylation and total protein levels of cells were assessed by Western blot analysis.Cell proliferation and apoptosis were examined by MTS assay, flow cytometry, and Western blots, respectively. ResultsCopanlisib more effectively inhibited cell proliferation in comparison to other PI3K inhibitors tested. HPV-positive HNSCC cells differentially responded to cisplatin, Afatinib, or Copanlisib. The combination of Afatinib and Copanlisib more effectively suppressed cell proliferation and induced apoptosis compared to either treatment alone. Mechanistically, the combination of Afatinib and Copanlisib completely blocked phosphorylation of EGFR, HER2, HER3, and Akt as well as significantly decreased the HPV E7 expression compared to either treatment alone. ConclusionAfatinib and Copanlisib more effectively suppress cell proliferation and survival of HPV-positive HNSCC in comparison to either treatment alone.