Genetic ablation of epidermal EGFR reveals the dynamic origin of adverse effects of anti-EGFR therapy

Abstract

Cancer patients treated with anti-EGFR (epidermal growth factor receptor) drugs often develop a dose-limiting pruritic rash of unknown etiology. The aims of our study were to define causal associations from a clinical study of cutaneous and systemic changes in patients treated with gefitinib and use these to develop and characterize a mouse model that recapitulates the human skin rash syndrome caused by anti-EGFR therapy. We examined the patients' plasma before and after treatment with gefitinib and documented changes in chemokines and leukocyte counts associated with the extent of rash or the presence of pruritus. We established a parallel mouse model by ablating EGFR in the epidermis. These mice developed skin lesions similar to the human rash. Before lesion development, we detected increased mRNA expression of chemokines in the skin associated with early infiltration of macrophages and mast cells and later infiltration of eosinophils, T cells, and neutrophils. As the skin phenotype evolved, changes in blood counts and circulating chemokines reproduced those seen in the gefitinib-treated patients. Crossing the mutant mice with mice deficient for tumor necrosis factor-α (TNF-α) receptors, MyD88, NOS2, CCR2, T cells, or B cells failed to reverse the skin phenotype. However, local depletion of macrophages provided partial resolution, suggesting that this model can identify targets that may be effective in preventing the troublesome and dose-limiting skin response to anti-EGFR drugs. These results highlight the importance of EGFR signaling in maintaining skin immune homeostasis and identify a macrophage contribution to a serious adverse consequence of cancer chemotherapy.

Fig.1

Inflammatory mediators and blood cell count changes in gefitinib-treated patients. Patients with ovarian cancer were treated with gefitinib (500 mg daily for 28 days) and matched plasma samples were studied before treatment (T=0) and after 1 cycle (T=1 month). Patients were characterized as having rash grade 1 (limited acneiform or maculopapular rash, limited or no symptoms; blue line) or grade 2 (more extensive rash and/or plaques, symptoms and/or requiring medical intervention; red line), and complaining of pruritus (red) or not (blue). Cytokine and chemokine concentrations were measured by ELISA-based multiplex assay. Associations of cytokine and chemokine concentrations (pg/ml) with rash (A) and pruritus (B) were analyzed. Blood cell count data were available for 8 of the 10 patients and were analyzed by calculating the percentage of granulocytes and lymphocytes present in the blood after one month of treatment minus the percentage before the treatment (Difference %) and correlating these differences with the extent of rash (C) and presence of pruritus (D). Changes in platelet count (absolute numbers k/μl) were reported as the number of platelets after treatment minus the number before treatment (D). P-values described in the text were calculated with a Wilcoxon rank sum test. Given the small number of patients and the large number of exploratory tests performed, p<0.01 was considered statistically significant, while 0.01 < p < 0.10 indicated trends.

Fig. 2

Consequences of epidermal ablation of EGFR in mice. Hematoxylin and eosin staining in skin sections and corresponding macroscopic phenotype of mice at different ages are shown at 3 days (A, B, C), 7 days (D, E, F), 21 days (G, H, I) and 4 months (J, K, L). Neutrophilic pustules (red arrows), keratin plugs (yellow arrows), sebaceous glands (green arrows) and pigment masses (black arrows) are highlighted in KO mouse skin sections and photographs (H, K, L). Objective 10X, scale bar 100 μm. Images were captured by staining skin sections from n > 3 independent litters.

Fig. 3

Systemic consequences of EGFR ablation in the epidermis. (A) Multiplex assay of plasma concentration of chemokines and cytokines expressed in pg/ml for 2, 3 and 12-week-old WT (white bars) and KO (black bars) mice. Graphs show mean pg/ml ± SD (n=3 individual readings, each from a pool of plasma from 6–7 mice/genotype/time point, * p≤0.05 by t test for KO compared to WT). Individual p values are listed in table S3. (B) Neutrophil (p=0.0001), platelet (p=0.0007) and lymphocyte (p=0.0008) mean counts ± SD in 3-week-old mice (blue WT and red KO, n=4 mice, by t test). (C) Spleen and lymph nodes from 2-month old WT and KO littermates are shown.

Fig. 4

Time course analysis of inflammatory infiltrate in EGFR-ablated mouse skin. (A, B) Immunohistochemical analysis of skin infiltrating cells was performed: F4/80 (macrophages), Toluidine blue (mast cells), CD3 (T cells in the dermis and gamma delta T cells in the epidermis), CD45R (B cells) and MBP (major basic protein/ eosinophils). The cells were counted in ten 200X fields at the indicated ages (A) for each mouse and expressed as the mean cell counts ± SD (n=3 mice, * p≤0.05 by t test, individual p values listed in table S3), WT white bars, KO black bars. (B) Representative 100X fields at day 7 in WT and KO mouse skin (scale bar 100 μm). (C) Myeloperoxidase (MPO) assay of skin biopsies isolated from the back of WT (blue) and KO (red) mice. The enzyme activity is monitored as changes in OD (optical density) over time. MPO activity is not detectable earlier than 2 weeks in both WT and KO mice. p=0.011 at 2 weeks, p=0.039 at 3 weeks. n=4 mice/time point/genotype, p calculated by t test. (D) MPO staining in sections from back skin and bone marrow of 3 month old WT and KO mice. Red arrows are pointing to microabscesses. Scale bars are 50 μm.

Fig.5

Time course of cytokine and chemokine changes in EGFR-ablated mouse skin. Sybr green based Real Time PCR analysis of cytokines, chemokines and chemokine receptors expressed over time after birth in mouse skin. Values are expressed as fold induction over WT values at 24 h, normalized to GAPDH. Mean expression ± SD, n=4 mice/genotype/time point, * p≤0.05 by t test of KO (black) versus corresponding WT (white), individual p values listed in table S3.

Fig. 6

Macrophage recruitment and hair phenotype in EGFR KO mice crossed with mice deficient for CCR2, B and T cells, MyD88, TNFR1/2, and NOS2. Representative F4/80 (left panels) and hematoxylin and eosin staining (right panels) in skin sections of TgK5Cre^(WT/+)/Egfr^(f/f)/CCR2^−/−, TgK5Cre^(WT/+)/Egfr^(f/f)/Rag1^−/−, TgK5Cre^(WT/+)/Egfr^(f/f)/MyD88^−/−, TgK5Cre^(WT/+)/Egfr^(f/f)/ TNFR1/2 ^−/−, all on a C57Bl/6 background, TgK5Cre^(WT/+)/Egfr^(f/f)/NOS2^−/− on a FVB/N background. All mice were 21 days old. Objective 10X, scale bar 100 μm. Images were captured by staining skin sections from n ≥ 3 independent litters per genotype.

Fig.7

Macrophage depletion and changes in KO skin phenotype and differentiation marker expression. (A) Hematoxylin and eosin staining in skin sections from WT and KO littermates treated with PBS liposomes or clodronate liposomes. Objective 20X, scale bar 100 μm. (B) Immunohistochemistry for F4/80 macrophages and Keratin 1 in mice treated as in (A). Objective 20X, scale bar 100 μm. Images were captured by staining skin sections from n=3 independent litters treated with clodronate injections.

Fig. 8

Differential effect of macrophage depletion on mRNA in EGFR KO mouse skin. (A, B, C) Sybr green based Real Time PCR analysis of chemokines, chemokine receptors, inflammatory mediators and epithelial differentiation markers. Values are expressed as percent change in clodronate treated KO compared to PBS-treated KO. (A, B) Values below zero on the y axis represent a decrease in KO mRNA levels after treatment with clodronate, with −100% corresponding to WT levels. (C) Changes with positive values (above zero on the y axis) represent a further increase in the mRNA expression in KO skin upon clodronate administration. No effect of clodronate is represented in values around zero. Experiments were performed on littermates from two litters of C57Bl/6 (black circles) and one litter of FVB/N mice (empty circles).