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Epidermal growth factor receptor mutation mediates cross-resistance to panitumumab and cetuximab in gastrointestinal cancer.

Braig F, März M, Schieferdecker A, Schulte A, Voigt M, Stein A, Grob T, Alawi M, Indenbirken D, Kriegs M, Engel E, Vanhoefer U, Grundhoff A, Loges S, Riecken K, Fehse B, Bokemeyer C, Binder M - Oncotarget (2015)

Bottom Line: It abrogates antibody binding and mediates cross-resistance to both antibodies in EGFR G465R-transfected Ba/F3 cells.In circulating tumor DNA from an independent "liquid biopsy" cohort of 27 patients, we found this novel mutation in 1 out of 6 panitumumab-treated cases while about one third of patients show acquired RAS mutations.We show that acquired resistance by epitope-changing mutations also emerges during panitumumab treatment, which can be easily detected by a liquid biopsy approach even before clinical resistance occurs and this may help in tailoring EGFR-targeted therapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology and Hematology, BMT with section Pneumology, Hubertus Wald Tumorzentrum / UCCH, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

ABSTRACT
Acquired resistance to epidermal growth factor receptor (EGFR) targeted antibodies represents a clinical challenge in the treatment of gastrointestinal tumors such as metastatic colorectal cancer, but its molecular mechanisms are incompletely understood. We scanned KRAS exon 2/3/4, NRAS exon 2/3/4 and the overlapping epitopes of the EGFR antibodies cetuximab and panitumumab for mutations in pre- and post-treatment tumor tissue of 21 patients with gastrointestinal cancer treated with chemotherapy +/- EGFR antibodies by next-generation sequencing ("tumor tissue" cohort). We describe a novel EGFR exon 12 mutation acquired in tumors of 1 out of 3 patients treated with panitumumab. The EGFR G465R mutation introduces a positive charge within the overlap of the panitumumab and cetuximab epitopes. It abrogates antibody binding and mediates cross-resistance to both antibodies in EGFR G465R-transfected Ba/F3 cells. In circulating tumor DNA from an independent "liquid biopsy" cohort of 27 patients, we found this novel mutation in 1 out of 6 panitumumab-treated cases while about one third of patients show acquired RAS mutations. We show that acquired resistance by epitope-changing mutations also emerges during panitumumab treatment, which can be easily detected by a liquid biopsy approach even before clinical resistance occurs and this may help in tailoring EGFR-targeted therapies.

No MeSH data available.


Related in: MedlinePlus

EGFR G465R mutation induces cross-resistance to panitumumab and cetuximab in an EGF-dependent Ba/F3 cellular modelA: EGFR signaling in EGF-dependent Ba/F3 model. Wt and S492R or G465R mutant EGFR-expressing Ba/F3 cells were cultured in the presence or absence of EGF and with addition of cetuximab, panitumumab, rituximab or erlotinib. After 2 hours, cells were harvested and EGFR/pEGFR expression analyzed by western blot analysis. B: Sensitivity of EGFR wt or EGFR G465R mutant-transfected Ba/F3 cells to treatment with EGFR-targeted antibodies. Ba/F3 cells were transformed to IL-3 independence with EGFR wt or mutant constructs and subsequently cultured in the presence or absence of EGF or with EGF in combination with panitumumab, cetuximab or control antibody rituximab. The number of viable cells was determined by trypan blue exclusion every 12 hours beginning 24 hours after seeding and plotted. Data are means from triplicate experiments +/−SEM.
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Figure 2: EGFR G465R mutation induces cross-resistance to panitumumab and cetuximab in an EGF-dependent Ba/F3 cellular modelA: EGFR signaling in EGF-dependent Ba/F3 model. Wt and S492R or G465R mutant EGFR-expressing Ba/F3 cells were cultured in the presence or absence of EGF and with addition of cetuximab, panitumumab, rituximab or erlotinib. After 2 hours, cells were harvested and EGFR/pEGFR expression analyzed by western blot analysis. B: Sensitivity of EGFR wt or EGFR G465R mutant-transfected Ba/F3 cells to treatment with EGFR-targeted antibodies. Ba/F3 cells were transformed to IL-3 independence with EGFR wt or mutant constructs and subsequently cultured in the presence or absence of EGF or with EGF in combination with panitumumab, cetuximab or control antibody rituximab. The number of viable cells was determined by trypan blue exclusion every 12 hours beginning 24 hours after seeding and plotted. Data are means from triplicate experiments +/−SEM.

Mentions: Next, we asked i) if receptor function was still preserved in the EGFR G465R mutant and ii) if the significant inhibition of antibody binding to EGFR G465R translated into resistance to panitumumab and cetuximab in a cellular model. To address this experimentally, we stably transfected murine EGFR-negative, IL-3-dependent Ba/F3 pro-B cells with the EGFR wt or mutant G465R and S492R constructs. After selection with G418, ectopic expression of wt and mutant EGFRin these cells conferred IL-3 independence in the presence of EGF, but not if erlotinib was added (Supplementary Figure 3). This indicated that EGF binding to the EGFR mutants was still preserved and receptor function intact. Stable Ba/F3 cell lines expressing wt or mutant EGFR were then treated with panitumumab, cetuximab, control antibody rituximab or erlotinib for 2 hours (Figure 2A). Whilst in EGFR wt cells cetuximab, panitumumab and erlotinib completely blocked EGFR phosphorylation, only panitumumab showed this effect in the S492R mutant. In the G465R mutant, none of the antibodies blocked EGFR phosphorylation. EGFR wt and G465R mutant Ba/F3 cells were then cultured for 108 hours in the presence of panitumumab, cetuximab or control antibody rituximab. Whilst EGFRwt transfected cells were sensitive to treatment with panitumumab and cetuximab, proliferation of EGFR G465R transfected cells was unaffected by treatment with either of these antibodies (Figure 2B). This data suggested that the G465R mutation, acquired under treatment with panitumumab, mediates cross-resistance to panitumumab and cetuximab by disrupting the antibody-EGFR interaction.


Epidermal growth factor receptor mutation mediates cross-resistance to panitumumab and cetuximab in gastrointestinal cancer.

Braig F, März M, Schieferdecker A, Schulte A, Voigt M, Stein A, Grob T, Alawi M, Indenbirken D, Kriegs M, Engel E, Vanhoefer U, Grundhoff A, Loges S, Riecken K, Fehse B, Bokemeyer C, Binder M - Oncotarget (2015)

EGFR G465R mutation induces cross-resistance to panitumumab and cetuximab in an EGF-dependent Ba/F3 cellular modelA: EGFR signaling in EGF-dependent Ba/F3 model. Wt and S492R or G465R mutant EGFR-expressing Ba/F3 cells were cultured in the presence or absence of EGF and with addition of cetuximab, panitumumab, rituximab or erlotinib. After 2 hours, cells were harvested and EGFR/pEGFR expression analyzed by western blot analysis. B: Sensitivity of EGFR wt or EGFR G465R mutant-transfected Ba/F3 cells to treatment with EGFR-targeted antibodies. Ba/F3 cells were transformed to IL-3 independence with EGFR wt or mutant constructs and subsequently cultured in the presence or absence of EGF or with EGF in combination with panitumumab, cetuximab or control antibody rituximab. The number of viable cells was determined by trypan blue exclusion every 12 hours beginning 24 hours after seeding and plotted. Data are means from triplicate experiments +/−SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494921&req=5

Figure 2: EGFR G465R mutation induces cross-resistance to panitumumab and cetuximab in an EGF-dependent Ba/F3 cellular modelA: EGFR signaling in EGF-dependent Ba/F3 model. Wt and S492R or G465R mutant EGFR-expressing Ba/F3 cells were cultured in the presence or absence of EGF and with addition of cetuximab, panitumumab, rituximab or erlotinib. After 2 hours, cells were harvested and EGFR/pEGFR expression analyzed by western blot analysis. B: Sensitivity of EGFR wt or EGFR G465R mutant-transfected Ba/F3 cells to treatment with EGFR-targeted antibodies. Ba/F3 cells were transformed to IL-3 independence with EGFR wt or mutant constructs and subsequently cultured in the presence or absence of EGF or with EGF in combination with panitumumab, cetuximab or control antibody rituximab. The number of viable cells was determined by trypan blue exclusion every 12 hours beginning 24 hours after seeding and plotted. Data are means from triplicate experiments +/−SEM.
Mentions: Next, we asked i) if receptor function was still preserved in the EGFR G465R mutant and ii) if the significant inhibition of antibody binding to EGFR G465R translated into resistance to panitumumab and cetuximab in a cellular model. To address this experimentally, we stably transfected murine EGFR-negative, IL-3-dependent Ba/F3 pro-B cells with the EGFR wt or mutant G465R and S492R constructs. After selection with G418, ectopic expression of wt and mutant EGFRin these cells conferred IL-3 independence in the presence of EGF, but not if erlotinib was added (Supplementary Figure 3). This indicated that EGF binding to the EGFR mutants was still preserved and receptor function intact. Stable Ba/F3 cell lines expressing wt or mutant EGFR were then treated with panitumumab, cetuximab, control antibody rituximab or erlotinib for 2 hours (Figure 2A). Whilst in EGFR wt cells cetuximab, panitumumab and erlotinib completely blocked EGFR phosphorylation, only panitumumab showed this effect in the S492R mutant. In the G465R mutant, none of the antibodies blocked EGFR phosphorylation. EGFR wt and G465R mutant Ba/F3 cells were then cultured for 108 hours in the presence of panitumumab, cetuximab or control antibody rituximab. Whilst EGFRwt transfected cells were sensitive to treatment with panitumumab and cetuximab, proliferation of EGFR G465R transfected cells was unaffected by treatment with either of these antibodies (Figure 2B). This data suggested that the G465R mutation, acquired under treatment with panitumumab, mediates cross-resistance to panitumumab and cetuximab by disrupting the antibody-EGFR interaction.

Bottom Line: It abrogates antibody binding and mediates cross-resistance to both antibodies in EGFR G465R-transfected Ba/F3 cells.In circulating tumor DNA from an independent "liquid biopsy" cohort of 27 patients, we found this novel mutation in 1 out of 6 panitumumab-treated cases while about one third of patients show acquired RAS mutations.We show that acquired resistance by epitope-changing mutations also emerges during panitumumab treatment, which can be easily detected by a liquid biopsy approach even before clinical resistance occurs and this may help in tailoring EGFR-targeted therapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology and Hematology, BMT with section Pneumology, Hubertus Wald Tumorzentrum / UCCH, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

ABSTRACT
Acquired resistance to epidermal growth factor receptor (EGFR) targeted antibodies represents a clinical challenge in the treatment of gastrointestinal tumors such as metastatic colorectal cancer, but its molecular mechanisms are incompletely understood. We scanned KRAS exon 2/3/4, NRAS exon 2/3/4 and the overlapping epitopes of the EGFR antibodies cetuximab and panitumumab for mutations in pre- and post-treatment tumor tissue of 21 patients with gastrointestinal cancer treated with chemotherapy +/- EGFR antibodies by next-generation sequencing ("tumor tissue" cohort). We describe a novel EGFR exon 12 mutation acquired in tumors of 1 out of 3 patients treated with panitumumab. The EGFR G465R mutation introduces a positive charge within the overlap of the panitumumab and cetuximab epitopes. It abrogates antibody binding and mediates cross-resistance to both antibodies in EGFR G465R-transfected Ba/F3 cells. In circulating tumor DNA from an independent "liquid biopsy" cohort of 27 patients, we found this novel mutation in 1 out of 6 panitumumab-treated cases while about one third of patients show acquired RAS mutations. We show that acquired resistance by epitope-changing mutations also emerges during panitumumab treatment, which can be easily detected by a liquid biopsy approach even before clinical resistance occurs and this may help in tailoring EGFR-targeted therapies.

No MeSH data available.


Related in: MedlinePlus