Antiestrogen resistance of breast cancer has been related to enhanced growth factor receptor expression and activation. We have previously shown that ectopic expression and subsequent activation of... Show moreAntiestrogen resistance of breast cancer has been related to enhanced growth factor receptor expression and activation. We have previously shown that ectopic expression and subsequent activation of the insulin-like growth factor-1 receptor (IGF1R) or the epidermal growth factor receptor (EGFR) in MCF7 or T47D breast cancer cells results in antiestrogen resistance. In order to identify novel therapeutic targets to prevent this antiestrogen resistance, we performed kinase inhibitor screens with 273 different inhibitors in MCF7 cells overexpressing IGF1R or EGFR. Kinase inhibitors that antagonized antiestrogen resistance but are not directly involved in IGF1R or EGFR signaling were prioritized for further analyses. Various ALK (anaplastic lymphoma receptor tyrosine kinase) inhibitors inhibited cell proliferation in IGF1R expressing cells under normal and antiestrogen resistance conditions by preventing IGF1R activation and subsequent downstream signaling; the ALK inhibitors did not affect EGFR signaling. On the other hand, MEK (mitogen-activated protein kinase kinase)1/2 inhibitors, including PD0325901, selumetinib, trametinib and TAK-733, selectively antagonized IGF1R signaling-mediated antiestrogen resistance but did not affect cell proliferation under normal growth conditions. RNAseq analysis revealed that MEK inhibitors PD0325901 and selumetinib drastically altered cell cycle progression and cell migration networks under IGF1R signaling-mediated antiestrogen resistance. In a group of 219 patients with metastasized ER + breast cancer, strong pMEK staining showed a significant correlation with no clinical benefit of first-line tamoxifen treatment. We propose a critical role for MEK activation in IGF1R signaling-mediated antiestrogen resistance and anticipate that dual-targeted therapy with a MEK inhibitor and antiestrogen could improve treatment outcome. Show less
Tan, D.S.W.; Kim, S.W.; Aix, S.P.; Sequist, L.V.; Smit, E.F.; Yang, J.C.H.; ... ; Kim, D.W. 2022
Introduction: Nazartinib, a novel third-generation EGFR-tyrosine kinase inhibitor, previously demonstrated antitumor activity and manageable safety in patients with EGFR-mut ant advanced non-small... Show moreIntroduction: Nazartinib, a novel third-generation EGFR-tyrosine kinase inhibitor, previously demonstrated antitumor activity and manageable safety in patients with EGFR-mut ant advanced non-small cell lung cancer (NSCLC) who received <= 3 prior lines of systemic ther-apy. Herein, we report phase 2 efficacy and safety of first-line nazartinib. Methods: This single-arm, open-label, global study enrolled treatment-naive adult patients with stage IIIB/IV NSCLC harboring EGFR-activating mutations (eg, L858R and/or ex19del). Patients with neurologically stable and controlled brain metastases were also eligible. Patients received oral nazartinib 150 mg once daily. The primary endpoint was Blinded Independent Review Committee (BIRC)-assessed overall response rate (ORR) per RECIST v1.1. Results: Forty-five patients received >= 1 dose of nazartinib. The median follow-up time from enrollment to data cutoff (November 1, 2019) was 30 months (range: 25-34). The BIRC-assessed ORR was 69% (95% CI, 53-82). The median progression-free survival (PFS) was 18 months (95% CI, 15-not estimable [NE]). The median overall survival was NE. In patients with baseline brain metastases (n = 18), the ORR and median PFS (95% CIs) were 67% (41-87) and 17 months (11-21). Seventeen of 18 patients had brain metastases as non-target lesions; the CNS lesions were absent/normalized in 9 of 17 (53%). Only 2 of 27 patients without baseline brain metastases developed new brain metastases postbaseline. Most frequent adverse events (>= 25%, any grade, all-causality) were diarrhea (47%), maculopapular rash (38%), pyrexia (29%), cough, and stomatitis (27% each). Conclusions: First-line nazartinib demonstrated promising efficacy, including clinically meaningful antitumor activity in the brain, and manageable safety in patients with EGFR-mutant NSCLC. (C) 2022 Published by Elsevier Ltd. Show less
This open-label Phase II study conducted prior to routine EGFR mutation testing, assessed whether acquired resistance to erlotinib in NSCLC patients with a given MET protein expression level... Show moreThis open-label Phase II study conducted prior to routine EGFR mutation testing, assessed whether acquired resistance to erlotinib in NSCLC patients with a given MET protein expression level enriched for EGFRmt could be overcome by emibetuzumab, an antibody against MET. Although some responses were seen, the trial did not meet its primary endpoint.Introduction: The hepatocyte growth factor receptor MET represents a resistance mechanism to epidermal growth factor receptor (EGFR) inhibition in EGFR mutant (mt) non-small cell lung cancer (NSCLC). This Phase 2 study tested whether acquired resistance to erlotinib in MET protein positive NSCLC patients enriched for EGFRmt can be overcome by emibetuzumab plus erlotinib. Patient and Methods: Patients with Stage IV NSCLC with acquired resistance to erlotinib and MET diagnostic (+) (>= 10% of cells expressing MET at >= 2+ IHC staining intensity at any time) were randomized (3:1) to receive emibetuzumab 750 mg every 2 weeks with or without erlotinib 150 mg once daily. The primary objective was to evaluate the overall response rate (ORR) relative to historic control, with a co-primary objective of ORR in patients with MET expression in >= 60% of cells >= 2+ (MET > 60%). Results: One hundred and eleven MET+ patients received emibetuzumab plus erlotinib (N = 83) or emibetuzumab monotherapy (N = 28). 89 of 111 MET+ samples were post-erlotinib. ORR was 3.0% for emibetuzumab plus erlotinib (95% CI: 0.4, 10.5) and 4.3% for emibetuzumab (95% CI: 0.1, 21.9), in patients with post-erlotinib progression biopsies available (n = 89). Similar results were observed in patients with MET >= 60% expression (n = 74). Disease control rate and progression-free survival were higher for emibetuzumab plus erlotinib (50%/3.3 months) than for emibetuzumab (26%/1.6 months). No unexpected safety signals emerged. Partial responses were observed in patients with and without EGFRmt or MET amplification. EGFR sensitizing mutations were identified retrospectively in 84.2% of those with available tissue (85/101). Conclusion: Acquired resistance to erlotinib in MET diagnostic (+) patients was not reversed by emibetuzumab plus erlotinib or emibetuzumab monotherapy, although a subset of patients obtained clinical benefit. (C) 2022 Elsevier Inc. All rights reserved. Show less
Background The epidermal growth factor receptor (EGFR) is a key protein involved in cancer development. Monoclonal antibodies targeting EGFR are approved for the treatment of metastatic colorectal... Show moreBackground The epidermal growth factor receptor (EGFR) is a key protein involved in cancer development. Monoclonal antibodies targeting EGFR are approved for the treatment of metastatic colorectal cancer (CRC). Despite the beneficial clinical effects observed in subgroups of patients, the acquisition of resistance to treatment remains a major concern. Protein N-glycosylation of cellular receptors is known to regulate physiological processes leading to activation of downstream signaling pathways. In the present study, the role of EGFR-specific terminal alpha 2,6-sialylation was analyzed in modulation of the malignant phenotype of CRC cells and their resistance to monoclonal antibody Cetuximab-based therapy.Methods Glycoengineered CRC cell models with specific sialyltransferase ST6GAL1 expression levels were applied to evaluate EGFR activation, cell surface glycosylation and therapeutic response to Cetuximab.Results Glycoproteomic analysis revealed EGFR as a major target of ST6Gal1-mediated alpha 2,6-sialylation in a glycosite-specific manner. Mechanistically, CRC cells with increased ST6Gal1 expression and displaying terminal alpha 2,6-sialylation showed a marked resistance to Cetuximab-induced cytotoxicity. Moreover, we found that this resistance was accompanied by downregulation of EGFR expression and its activation.Conclusions Our data indicate that EGFR alpha 2,6-sialylation is a key factor in modulating the susceptibility of CRC cells to antibody targeted therapy, thereby disclosing a potential novel biomarker and providing key molecular information for tailor made anti-cancer strategies. Show less
Simple SummaryNear-infrared imaging of tumors during surgery facilitates the oncologic surgeon to distinguish malignant from healthy tissue. The technique is based on fluorescent tracers binding to... Show moreSimple SummaryNear-infrared imaging of tumors during surgery facilitates the oncologic surgeon to distinguish malignant from healthy tissue. The technique is based on fluorescent tracers binding to tumor biomarkers on malignant cells. Currently, there are no clinically available fluorescent tracers that specifically target soft tissue sarcomas. This review searched the literature to find candidate biomarkers for soft tissue sarcomas, based on clinically used therapeutic antibodies. The search revealed 7 biomarkers: TEM1, VEGFR-1, EGFR, VEGFR-2, IGF-1R, PDGFR alpha, and CD40. These biomarkers are abundantly present on soft tissue sarcoma tumor cells and are already being targeted with humanized monoclonal antibodies. The conjugation of these antibodies with a fluorescent dye will yield in specific tracers for image-guided surgery of soft tissue sarcomas to improve the success rates of tumor resections.Surgery is the mainstay of treatment for localized soft tissue sarcomas (STS). The curative treatment highly depends on complete tumor resection, as positive margins are associated with local recurrence (LR) and prognosis. However, determining the tumor margin during surgery is challenging. Real-time tumor-specific imaging can facilitate complete resection by visualizing tumor tissue during surgery. Unfortunately, STS specific tracers are presently not clinically available. In this review, STS-associated cell surface-expressed biomarkers, which are currently already clinically targeted with monoclonal antibodies for therapeutic purposes, are evaluated for their use in near-infrared fluorescence (NIRF) imaging of STS. Clinically targeted biomarkers in STS were extracted from clinical trial registers and a PubMed search was performed. Data on biomarker characteristics, sample size, percentage of biomarker-positive STS samples, pattern of biomarker expression, biomarker internalization features, and previous applications of the biomarker in imaging were extracted. The biomarkers were ranked utilizing a previously described scoring system. Eleven cell surface-expressed biomarkers were identified from which 7 were selected as potential biomarkers for NIRF imaging: TEM1, VEGFR-1, EGFR, VEGFR-2, IGF-1R, PDGFR alpha, and CD40. Promising biomarkers in common and aggressive STS subtypes are TEM1 for myxofibrosarcoma, TEM1, and PDGFR alpha for undifferentiated soft tissue sarcoma and EGFR for synovial sarcoma. Show less
Verheijen, R.B.; Duijl, T.T. van; Heuvel, M.M. van den; Vessies, D.; Muller, M.; Beijnen, J.H.; ... ; Huitema, A.D.R. 2021
Purpose We studied EGFR mutations in circulating tumor DNA (ctDNA) and explored their role in predicting the progression-free survival (PFS) of non-small cell lung cancer (NSCLC) patients treated... Show morePurpose We studied EGFR mutations in circulating tumor DNA (ctDNA) and explored their role in predicting the progression-free survival (PFS) of non-small cell lung cancer (NSCLC) patients treated with erlotinib or gefitinib. Methods The L858R, T790M mutations and exon 19 deletions were quantified in plasma using digital droplet polymerase chain reaction (ddPCR). The dynamics of ctDNA mutations over time and relationships with PFS were explored. Results In total, 249 plasma samples (1-13 per patient) were available from 68 NSCLC patients. The T790M and L858R or exon 19 deletion were found in the ctDNA of 49 and 56% patients, respectively. The median (range) concentration in these samples were 7.3 (5.1-3688.7), 11.7 (5.1-12,393.3) and 27.9 (5.9-2896.7) copies/mL, respectively. Using local polynomial regression, the number of copies of EGFR mutations per mL increased several months prior to progression on standard response evaluation. Conclusion This change was more pronounced for the driver mutations than for the resistance mutations. In conclusion, quantification of EGFR mutations in plasma ctDNA was predictive of treatment outcomes in NSCLC patients. In particular, an increase in driver mutation copy number could predict disease progression. Show less
Introduction: Access to targeted therapies for lung cancer depends on the accurate identification of patients' biomarkers through molecular testing. The International Association for the Study of... Show moreIntroduction: Access to targeted therapies for lung cancer depends on the accurate identification of patients' biomarkers through molecular testing. The International Association for the Study of Lung Cancer (IASLC) conducted an international survey to evaluate perceptions on current practice and barriers to implementation of molecular testing.Methods: We distributed the survey to IASLC members and other health care professionals around the world. The survey included a seven-question introduction for all respondents, who then answered according to one of three tracks: (1) requesting tests and treating patients, (2) performing and interpreting assays, or (3) tissue acquisition. Barriers to implementing molecular testing were provided in free-response fields. The chi-square test was used for regional comparisons.Results: A total of 2537 respondents from 102 countries participated. Most respondents who test and treat patients believe that less than 50% of patients with lung cancer in their country receive molecular testing, but reported higher rates within their own practice. Although many results varied by region, the five most frequent barriers cited in all regions were cost, quality and standards, access, awareness, and turnaround time. Many respondents expressed dissatisfaction with the current state of molecular testing for lung cancer, including 41% of those performing and interpreting assays. Issues identified included trouble understanding results (37%) and the quality of the samples (23% reported >10% rejection rate). Despite concerns regarding the quality of testing, 47% in the performing and interpreting track stated there is no policy or strategy to improve quality in their country. In addition, 33% of respondents who request tests and treat patients were unaware of the most recent College of American Pathologists, IASLC, and Association for Molecular Pathology guidelines for molecular testing.Conclusions: Adoption of molecular testing for lung cancer is relatively low across the world. Barriers include cost, access, quality, turnaround time, and lack of awareness. (c) 2020 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved. Show less
Targeted photodynamic therapy (PDT) has the potential to selectively damage tumor tissue and to increase tumor vessel permeability. Here we characterize the tissue biodistribution of two EGFR... Show moreTargeted photodynamic therapy (PDT) has the potential to selectively damage tumor tissue and to increase tumor vessel permeability. Here we characterize the tissue biodistribution of two EGFR-targeted nanobody-photosensitizer conjugates (NB-PS), the monovalent 7D12-PS and the biparatopic 7D12-9G8-PS. In addition, we report on the local and acute phototoxic effects triggered by illumination of these NB-PS which have previously shown to lead to extensive tumor damage.Methods: Intravital microscopy and the skin-fold chamber model, containing OSC-19-luc2-cGFP tumors, were used to investigate: a) the fluorescence kinetics and distribution, b) the vascular response and c) the induction of necrosis after illumination at 1 or 24 h post administration of 7D12-PS and 7D12-9G8-PS. In addition, dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) of a solid tumor model was used to investigate the microvascular status 2 h after 7D12-PS mediated PDT.Results: Image analysis showed significant tumor colocalization for both NB-PS which was higher for 7D12-9G8-PS. Intravital imaging showed clear tumor cell membrane localization 1 and 2 h after administration of 7D12-9G8-PS, and fluorescence in or close to endothelial cells in normal tissue for both NB-PS. PDT lead to vasoconstriction and leakage of tumor and normal tissue vessels in the skin-fold chamber model. DCE-MRI confirmed the reduction of tumor perfusion after 7D12-PS mediated PDT. PDT induced extensive tumor necrosis and moderate normal tissue damage, which was similar for both NB-PS conjugates. This was significantly reduced when illumination was performed at 24 h compared to 1 h after administration.Discussion: Although differences were observed in distribution of the two NB-PS conjugates, both led to similar necrosis. Clearly, the response to PDT using NB-PS conjugates is the result of a complex mixture of tumor cell responses and vascular effects, which is likely to be necessary for a maximally effective treatment. Show less
Endocytosis is a crucial mechanism for the cell to maintain homeostasis. By vesicular transport the cell is able to take up cargo from the cell surface, send molecules to the plasma membrane for... Show moreEndocytosis is a crucial mechanism for the cell to maintain homeostasis. By vesicular transport the cell is able to take up cargo from the cell surface, send molecules to the plasma membrane for excretion or to regulate positioning of internalized cargo in the cell. This thesis describes the regulation of transport complexes associated to these vesicles that determines their movement in the cell. Through recruitment of RAB7GAP the GTPase Rab7 can be removed from the vesicular membrane, allowing for transport mediated by another GTPase called Arl8b.The second part of this thesis focusses on the bacterium Salmonella which manipulates the endosomal system in order to create a niche that supports its own survival. By excretion of effector molecules Salmonella imodulates the membrane content of vesicles and thereby prevents its own lysosomal degradation. This allows Salmonella to create a Salmonella containing vesicle compartment that benefits its own survival in the host cell. This thesis describes how release of these effector molecules can support transformation of host cells. These results explain the strrong correlation between gallbladder cancer and chronic S. Typhi infections observed in patients from countries like India, where S. Typhi infections are still common. Show less
The research described in this thesis focused on identifying novel drug targets and synergistic combinations for triple-negative breast cancer (TNBC), a virulent subtype of breast cancer with a... Show moreThe research described in this thesis focused on identifying novel drug targets and synergistic combinations for triple-negative breast cancer (TNBC), a virulent subtype of breast cancer with a dismal prognosis and limited therapeutic options. In particular, the work centred on reversing resistance of TNBC cells to EGFR inhibitors. High-throughput kinase inhibitor library-based screens were utilised to evaluate the potential of novel targeted agents in a panel of TNBC cell lines and subsequently identify TNBC-specific genetic dependencies using siRNA-based screening. The signal transduction pathways perturbed by drug treatment were delineated and subsequently scrutinised using transcriptomic profiling and western blotting. The impact of drug treatment or gene silencing on cell death, proliferation, cell cycle progression and migration was assessed simultaneously. This work demonstrated that TNBC cell lines resistant to both MEK and Akt inhibitors are sensitive to disruption of CDK function. Additionally, it revealed that novel CDK inhibitors with strong activity against P-TEFb/CDK9 are highly effective against TNBC cells as single agents and in combination with multiple targeted therapies. These agents provoked profound down-regulation of multiple oncogenic pro-proliferative pathways, the silencing of which was detrimental to TNBC cell proliferation, thus defining several genes as potential future drug targets. Show less
Bakker, J.; Spits, M.; Neefjes, J.; Berlin, I. 2017
Pathology laboratories throughout the world have compiled large archives of unique collections of tissue specimens. These tissue samples are used for patient diagnostics and research. Novel... Show morePathology laboratories throughout the world have compiled large archives of unique collections of tissue specimens. These tissue samples are used for patient diagnostics and research. Novel molecular insights into alterations in normal cellular function have led to the identification of targets for innovative therapies. Testing for biomarkers combined with molecular pathology has created the potential for __personalized medicine__ and improved diagnosis, treatment and prognosis. New technologies for molecular analysis in molecular tumor diagnostics and research must be developed and implemented to keep pace with the latest insights, resulting in a constant cycle of change. Such translational research can only progress if patient material can be accessed from the archives for further study. The resulting new insights and strategies will eventually be implemented in patient care. This thesis describes three important issues in this cycle of change with a focus on molecular pathology. Tissue preparation, method development and data analysis. Show less
